20- HETE Signals Through G Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular
Function and Trigger Hypertension
Victor Garcia1, Ankit Gilani1, Brian Shkolnik1, Varunkumar Pandey1, Frank Fan Zhang1, Rambabu
Dakarapu2, Shyam K. Gandham2, N. Rami Reddy2, Joan P. Graves3, Artiom Gruzdev3, Darryl C. Zeldin3,
Jorge H. Capdevila4, John R. Falck2 and Michal Laniado Schwartzman1
1
Department of Pharmacology, New York Medical College School of Medicine, Valhalla, NY 10595;
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390,
3
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, and
4
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37240.
2
Running title: 20-HETE-GPR75 Pairing and Hypertension
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Subject Terms:
Vascular Biology
Hypertension
Cell Signaling/Signal Transduction
Remodeling
Address correspondence to:
Dr. Michal Laniado Schwartzman
Department of Pharmacology
New York Medical College School of Medicine
15 Dana Road
Valhalla, NY 10595
Tel: (914) 594-3116
Fax: (914) 347-4956
[email protected]
In February 2016, the average time from submission to first decision for all original research papers
submitted to Circulation Research was 15.4 days.
DOI: 10.1161/CIRCRESAHA.116.310525 1
ABSTRACT
Rationale: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 (CYP)
eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle
contractility, migration and proliferation, as well as endothelial cell dysfunction and inflammation.
Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension,
stroke, myocardial infarction and vascular diseases.
Objective: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific
agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE
binds and through which it activates a signaling cascade that culminates in many of the functional
outcomes attributed to 20-HETE in vitro and in vivo.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Methods and Results: Using crosslinking analogs, click chemistry, binding assays, and functional assays,
we identified GPR75, currently an orphan G-protein coupled receptor (GPCR), as a specific target of 20HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein
dissociation and increased inositol phosphate (IP-1) accumulation as well as GPCR-kinase interacting
protein-1 (GIT1)-GPR75 binding, which further facilitated the c-Src-mediated transactivation of
endothelial EGFR. This results in downstream signaling pathways which induce angiotensin-converting
enzyme (ACE) expression and endothelial dysfunction. Knockdown of GPR75 or GIT1 prevented 20HETE-mediated endothelial growth factor receptor (EGFR) phosphorylation and ACE induction. In
vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GIT1-mediated
protein kinase C (PKC)-stimulated phosphorylation of MaxiKλ , linking GPR75 activation to 20-HETEmediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension
prevented blood pressure elevation and 20-HETE-mediated increases in ACE expression, endothelial
dysfunction, smooth muscle contractility and vascular remodeling.
Conclusions: This is the first report to identify a GPCR target for an eicosanoid of this class. The
discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and
pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.
Keywords:
20-HETE, GPR75, GIT1, EGFR, ACE, Vascular Remodeling, hypertension,cytochrome P450/eicosanoids.
DOI: 10.1161/CIRCRESAHA.116.310525 2
Nonstandard Abbreviations and Acronyms:
20-HETE
CYP
EGFR
eNOS
IKK
NFB
MAPK
ACE
Ang II
GPCR
GPR75
20-APheDa
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
12-HETE
20-HEDE
DBCO
TGFB1I1
HIC-5
GIT1
NO
IP-1
PKC
DOX
MaxiK
20-hydroxyeicosatetraenoic acid
Cytochrome P450
Epidermal Growth Factor Receptor
Endothelial Nitric Oxide Synthase
Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Beta
Nuclear Factor Kappa B
Mitogen activated protein kinase
Angiotensin Converting Enzyme
Angiotensin II
G-Protein Coupled Receptor
G-Protein Receptor 75
20-azido-N-((4-(3-(4-benzoylphenyl)propanamido)-phenyl)sulfonyl)-19(S)hydroxyeicosa-5(Z),14(Z)-dienamide
12-Hydroxyeicosatetraenoic Acid
20-hydroxyeicosa-6(Z),15(Z)-dienoic acid
Dibenzocyclooctyne
Transforming Growth Factor Beta-1-Induced Transcript 1
Hydrogen Peroxide-Inducible Clone 5
G Protein-Coupled Receptor Kinase Interactor 1
Nitric Oxide
Inositol Phosphate
Protein Kinase C
Doxycycline
Large Conductance Voltage and Calcium-Activated Potassium Subunit Beta
DOI: 10.1161/CIRCRESAHA.116.310525 3
INTRODUCTION
Hypertension is the leading cause of stroke and cardiovascular diseases and the chief risk factor
for global disease burden. With few exceptions, the molecular bases of the most common forms of human
hypertension are yet to be defined and, thus, its early diagnosis and clinical management remains
challenging, reflecting the complexity of a disease in which multiple environmental and genetic factors
contribute to its multifaceted etiology.1 Studies from us and others, added 20-hydroxyeicosatetraenoic
acid (20-HETE), the product of the ω-hydroxylation of arachidonic acid by cytochrome P450 (CYP) 4A
and 4F isozymes,2 to the list of factors contributing to the pathophysiology of hypertension and its
cardiovascular consequences.3-5 This is supported by studies identifying associations between variants in
the human CYP4A11 and CYP4F2 genes and the prevalence of hypertension, myocardial infarction and
stroke,6-9 and clinical studies showing changes in plasma and urinary levels of 20-HETE in hypertension
as well as in diseases and conditions such as cancer,10 endothelial dysfunction,11 oxidative stress,12 obesity
and metabolic syndrome,13, 14 diabetes,15 autosomal dominant polycystic kidney disease16 and chronic
kidney disease.17, 18
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
The proposed contributions of 20-HETE to the etiology of cardiovascular diseases stem primarily
from its reported actions on the vasculature. 20-HETE is a potent vasoactive eicosanoid whose vascular
effects include stimulation of smooth muscle contractility, migration and proliferation, as well as
endothelial cell dysfunction and inflammation.19, 20 While studying 20-HETE-triggered signaling cascades
mediating endothelial dysfunction and activation, we identified the phosphorylation of the epidermal
growth factor receptor (EGFR) as a first step in a MAPK-IKK-NFB transduction pathway leading to
endothelial nitric oxide synthase (eNOS) uncoupling, inflammatory cytokine production and to increases
in angiotensin-converting enzyme (ACE) expression and activity.21-24 Though the mechanisms by which
20-HETE activates EGFR phosphorylation remained unknown, we postulated that 20-HETE could either:
a) cross the cell membrane and stimulate tyrosine kinases within the intracellular milieu, or b) interact
with a G-protein coupled receptor (GPCR), the activation of which could lead to transactivation of the
EGFR, as was documented for many autacoids including Angiotensin II (Ang II). 25, 26 Interestingly,
Akbulut et al., 27 showed that 20-HETE activates the Raf/MEK/ERK pathway in renal epithelial cells
through an EGFR- and c-Src-dependent mechanism and proposed the presence of a 20-HETE-specific
GPCR-EGFR transactivation through c-Src. The presence of a receptor/binding site for 20-HETE was
suggested by the development of specific analogs and antagonists that mimic or block many of the known
20-HETE bioactions.22, 28 With this in mind, we embarked on the challenging task of searching for the
“20-HETE Receptor”. Using crosslinking analogs, click chemistry, proteomics, protein partner analysis,
receptor binding assays and gene silencing we identified G-protein receptor 75 (GPR75), currently an
orphan GPCR, as the 20-HETE receptor capable of binding 20-HETE with high affinity and of activating
a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro
and in vivo.
DOI: 10.1161/CIRCRESAHA.116.310525 4
METHODS
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Expanded methods are presented in the Online Data Supplement. Primary (passages 2-3) human
microvascular endothelial cells (Invitrogen, Carlsbad, CA) and the rat aortic vascular smooth muscle cell
line A7r5 (ATCC, Manassas, VA) were used for all in vitro experiments. Click=in chemistry and binding
studies were conducted using endothelial cell membranes. Pull-down experiments and western blot
analysis of GPR75 was performed using a polyclonal antibody against the c-terminal (sc-164538, Santa
Cruz, Biotechnology, Dallas, TX) which recognized a single 54-55 KDa protein band. All experimental
protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and by the
Institutional Biosafety Committee. The generation and phenotypic characterization of the Cyp4a12
transgenic mice in which the expression of the Cyp4a12-20-HETE synthase is under the control of a
tetracycline (doxycycline, DOX)-sensitive promoter have been previously described. 29 Male Cyp4a12tg
mice (8-14-week-old) were used in all experiments. Mice were maintained on a 12 h light/dark cycle and
fed ad libitum. DOX (1 mg/ml) was administered in the drinking water. In some experiments, Cyp4a12tg
mice were anesthetized under isoflurane and administered lentiviral constructs via a single retro-orbital
injection to maximize systemic distribution. Each mouse was injected with 100 µl of either 4.33x109
TU/mL of non-targeting or 7.29x109 TU/mL of GPR75-targeted shRNA particles. The administration of
DOX (1 mg/ml) was initiated 48 h after the lentiviral injection. Blood pressure was monitored by
radiotelemetry and tail cuff before and after administration of DOX for 14-35 days. At the end of all
experiments, mice were anesthetized and laporotomy was performed. Renal preglomerular arteries (the
whole circulatory tree) or interlobar arteries (80-100µm) were microdissected and collected for western
blot analysis and functional studies. All results are expressed as mean ± SEM. Significance of difference
in mean values was determined using t test and 1-way ANOVA, followed by the Newman-Keul post hoc
test. P<0.05 was considered to be significant.
RESULTS
Identification of GPR75-20-HETE pairing.
We synthesized an analog of 20-HETE, 20-azido-N-((4-(3-(4-benzoylphenyl)propanamido)phenyl)sulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide (20-APheDa) (Fig. 1A), that contained both
benzophenone, a photoreactive crosslinker for protein binding, and an azide, for selective binding and
labeling to a click-chemistry dibenzocyclooctyne (DBCO) 800CW Infrared Dye. 20-APheDa functions as
a 20-HETE antagonist as inferred from its ability to block 20-HETE-mediated sensitization of
phenylephrine vasoconstriction by decreasing (4-fold) the EC50 (Fig. 1B). Incubation of 10 nmol/L of
20-APheDa with membrane fractions of human endothelial cells (EC) (20μg) followed by 15 min of UV
(365 nm) crosslinking and 1h of incubation with the click reagent (DBCO-IRdye 800CW, LiCor) (50
µmol/L) yielded several bands, including a dominant band located at 47-49 kDa were detected (Fig. 1C).
This labeling profile of several bands is characteristic of many click-chemistry interactions whereby
multiple proteins in close proximity to the binding site of the compound, in this case 20-APheda, are
labeled.30 Nevertheless, the labeling of these bands by 20-APheDa (0.1 nmol/L) was competed by excess
amounts of 20-HETE (100 μmol/L), but not 12-HETE (100 μmol/L) (Fig. 1D-E).
In-gel 20-APheDa-protein complexes were extracted from independent incubations and protein
profiling by Applied Biomics (Hayward, CA). Protein identification of the dominant band location was
based on peptide fingerprint mass mapping (using MS data) and peptide fragmentation mapping (using
MS/MS data). The MASCOT search engine was used to identify proteins from primary sequence
databases. Analysis of sequenced samples from the dominant band location identified several proteins and
domains as top hits including transforming growth factor beta-1-induced transcript 1 or hydrogen
DOI: 10.1161/CIRCRESAHA.116.310525 5
peroxide-inducible clone 5 (TGFB1I1/HIC-5),31 POTE ankyrin domain family members, Zinc finger
proteins, and GTP-binding proteins as top hits (Online Figure I). Further analysis also suggested domains
and proteins associated with GIT1, a GPCR-kinase interacting protein-1 scaffold protein with ADPribosylating factor GTPase activity providing clues towards the possibility of a multi-protein receptor
complex in close proximity to 20-APheDa's binding site.32 With the identification of these two particular
proteins we utilized protein partner analysis against orphan Gq Class A receptors referencing STRING
and NCBI protein partner databases. The analysis revealed an association of HIC-5 and GIT1 with a
candidate orphan receptor GPR75. To this end, in one out of three separate experiments GPR75-specific
sequences were detected with C.I. of 99.045%, based on peptide fingerprint mass and fragmentation
mapping using the MASCOT search engine, encouraging further investigation as to a potential
relationship between 20-HETE and GPR75. Moreover, databases including the Human Protein Atlas,
BioGPS and GeneAtlas demonstrate tissue-specific expression of Gpr75 to be broadly distributed across
various organs including the brain, endocrine tissues, lung, kidney, heart, adipose tissues, aorta and other
tissues. Our assessment of Gpr75 expression in tissues from C57BL/6 mice by qPCR concurred with the
distribution profile presented in these databases (Online Figure II). Many of these tissues are heavily
vascularized and have the capacity to produce 20-HETE.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Analyses of competition binding studies were performed. As seen in Figure 2A, 20-HETE but not
12(S)-HETE displaced bound [3H8] 20-HETE (6.67 nmol/L) from EC membranes in a concentrationdependent manner with a calculated Kd of 3.75 nmol/L (Fig. 2B). Knockdown of GPR75 (Fig. 2C)
prevented 3H-20-HETE binding to EC membranes (Fig. 2D). Likewise, addition of the 20-HETE
antagonist 20-5,16-HEDGE inhibited 3H-20-HETE binding to EC membranes (Fig. 2F). This experiment
confirmed our initial target protein identification (Online Figure I). Furthermore, the Kd values in these
experiments (Fig 2B and E) accurately represent 20-HETE’s biological activities wherein maximal
changes in nitric oxide (NO) bioavailability and ACE expression have been seen in the range of 1-10
nmol/L.22-24 Since GPR75 is a Gq-coupled receptor, the ability of 20-HETE to increase inositol
phosphate (IP-1) accumulation in EC as an index of its activation was further assessed. Exposure of EC
to 20-HETE (10 nmol/L) for 5 min increased IP-1 accumulation by 4-fold; this increase was not observed
in GPR75-knockdowned EC (Fig 2G-H). All together, these results show that 20-HETE binds to EC
membranes, pairs with GPR75 in a specific manner and functionally activates the receptor.
GPR75 is expressed in the vascular endothelium and is associated with G
q/11,
GIT1 and HIC-5.
Antibodies against the GPR75 c-terminal, which recognize a single 54-55 KDa protein band
(Online Figure III), were used in all subsequent experiments. A representative image of kidney sections
co-stained with antibodies against GPR75 and CD31, an endothelial cell marker, showed a clear colocalization of GPR75 to the vascular endothelium and expression of GPR75 can be observed throughout
the vessel (Fig. 3A). Moreover, immunoprecipitation and immunoblotting experiments indicated that
Gq/11 as well as G Protein-Coupled Receptor Kinase Interactor 1 (GIT1) and Hydrogen PeroxideInducible Clone 5 (HIC-5) are associated with GPR75 in EC (Fig. 3B).
20-HETE alters the association of GPR75 with Gq/11, GIT1 and HIC-5 in HMVEC.
Incubation of EC with 20-HETE (5 min) decreased GPR75-Gq/11 association by 44 and 51% at 1
and 10 nmol/L (Fig. 3C). In addition, 20-HETE also increased GPR75-GIT1 binding by 2-fold and HIC5-GPR75 dissociation by 60% (Fig. 3D-E). Importantly, 20-6,15-HEDGE, an effective 20-HETE
antagonist in vitro and in vivo,29, 33 did not increase GIT1-GPR75 association. Moreover, it prevented 20HETE-stimulated GIT1-GPR75 association (Fig. 3F).
DOI: 10.1161/CIRCRESAHA.116.310525 6
GPR75 and GIT1 are required for 20-HETE-mediated EGFR phosphorylation and downstream
signaling.
Previously, we documented that phosphorylation of EGFR is an early step in 20-HETE-mediated
activation of a MAPK-IKK-NFB signaling leading to eNOS uncoupling, ACE induction and
inflammatory cytokine production in EC. 22, 24, 34 To investigate the role of GPR75 and GIT1 in 20-HETEmediated phosphorylation of EGFR, small interfering RNAs (siRNA) against GPR75 and GIT1 were
used. Transfection of EC with siRNAs against GPR75 and GIT1 produced a maximal knockdown of 85
and 70%, respectively (Fig. 4A-B). As previously shown,24 20-HETE increases total tyrosine
phosphorylation of immunoprecipitated EGFR in EC transduced with control siRNA by 1.99±0.16-fold
(Fig. 4C). 20-HETE-stimulated EGFR tyrosine phosphorylation was completely prevented in EC
transduced with either GPR75 or GIT1 siRNA (Fig. 4C). In addition, within the same time frame in
which 20-HETE stimulated a 2.3-fold increase in EGFR phosphorylation (Fig. 4D), it also decreased the
association of c-Src with GIT1 by 72% (Fig. 4E) and increased association of c-Src with EGFR by 110%
(Fig. 4F), suggesting that 20-HETE binding to GPR75 activates a c-SRC-mediated EGFR
phosphorylation via GIT1.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
20-HETE-mediated induction of ACE mRNA requires GPR75.
One of the most prominent effects of 20-HETE in the vascular endothelium is induction of ACE
transcription and activity.22, 24 Here, we show that suppression of GPR75 in EC by GPR75-specific
siRNAs negated the 3.4-fold induction of ACE mRNA by 20-HETE (Fig. 4G), indicating that GPR75-20HETE pairing is a necessary step for 20-HETE-mediated induction of ACE. Importantly, the chemokine
CCL5, a proposed GPR75 ligand, 35 did not induce ACE transcription nor did it increase EGFR tyrosine
phosphorylation or Gq/11-GPR75 association in EC (Online Figure IV).
GPR75 knockdown prevents 20-HETE-dependent hypertension, vascular dysfunction and remodeling.
The Cyp4a12 transgenic mice (Cyp4a12tg) in which the expression of the Cyp4a12-20-HETE
synthase is under the control of doxycycline (DOX) display, upon administration of DOX, increased
levels of 20-HETE that is associated with vascular dysfunction and hypertension, both of which are
prevented or reversed by inhibiting the biosynthesis or blocking the actions of 20-HETE.29, 33 We used
this model to assess whether GPR75 is necessary for the pro-hypertensive actions of 20-HETE. Mice
were given a bolus injection of either control or GPR75-targeted shRNA lentiviral particles into the
retroorbital sinus followed by administration of DOX in the drinking water to induce Cyp4a12-20-HETE
synthase. As expected, administration of DOX to Cyp4a12tg mice that received a bolus of control shRNA
lentiviral particles or its vehicle resulted in a rapid and sustained increase in systolic blood pressure
measured by the tail cuff method (135±2 and 131±3 mmHg, respectively) (Fig. 5A-B). In contrast, DOX
administration to Cyp4a12tg mice that received a bolus of GPR75-targeted shRNA failed to increase
blood pressure (110±2 mmHg) (tail cuff, Fig. 5A-B & telemetry, online Figure V). Western blot analysis
of renal preglomerular microvessels from mice receiving GPR75-targeted shRNA lentiviral particles
confirmed an 80% knockdown of GPR75 levels (Fig. 5C). Similar reduction in GPR75 expression was
seen in other tissues, including the liver (45%) and heart (80%) (Online Figure VI). Elevated vascular
ACE has been characterized as a hallmark of the DOX-induced 20-HETE-dependent hypertension in
Cyp4a12tg mice. 33 In line with previous studies, vascular ACE expression in DOX- and DOX+control
shRNA-treated Cyp4a12tg mice increased by 3.3- and 3.8-fold, respectively (Fig. 5D). In contrast,
vascular ACE expression was not induced in DOX-treated Cyp4a12tg mice that received GPR75-targeted
shRNA lentiviral particles (Fig. 5D), further substantiating the notion that the GPR75-20-HETE pairing is
critical to achieve 20-HETE-mediated induction of ACE.
DOI: 10.1161/CIRCRESAHA.116.310525 7
The hypertensive phenotype of the DOX-treated Cyp4a12tg mice is associated with 20-HETEdependent endothelial dysfunction and enhanced sensitivity to constrictor stimuli.29, 33 Here we show that
knockdown of GPR75 interferes with the ability of DOX to impair relaxations to acetylcholine and
increase contractions to phenylephrine. The relaxation to acetylcholine was markedly reduced in
interlobar arteries from DOX-treated (55%±3%) as compared to arteries from water-treated Cyp4a12tg
mice (99±2%). Administration of GPR75-targeted, but not control, shRNA lentiviral particles prevented
DOX-induced impairment in relaxation to acetylcholine (88%±3%) (Fig. 6A). Likewise, administration of
GPR75-targeted shRNA prevented the DOX-induced increases in contractions to phenylephrine.
Treatment with DOX increased sensitivity to phenylephrine (p<0.05) as evidenced by a reduction in EC50
(from 0.75±011 to 0.40±0.08 µmol/L) and an increase in Rmax (from 4.89±0.51 to 6.17±0.39 mN/mm)
when compared to water-treated Cyp4a12tg mice (Fig. 6B). However, the EC50 to phenylephrine in
arteries from DOX-treated mice that received GPR75-targeted shRNA was unchanged (0.86±0.24
µmol/L) and was not different from the EC50 in arteries from control mice (Fig. 6B).
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Remodeling of the renal microvasculature is a striking pathology associated with chronic
hypertension. Prolonged exposure of the vasculature to high levels of 20-HETE as in the androgen-treated
rats or Cyp4a12tg mice receiving DOX leads to hypertrophic remodeling of the renal microvessels in a
20-HETE-dependent manner that is largely independent of blood pressure elevation.33, 36 In this study,
assessment of remodeling in interlobar arteries (~80-100 µm) showed that hypertension in Cyp4a12tg
mice receiving control shRNA+DOX for 35 days was associated with a 3-fold increase in media
thickness, media to lumen ratio and cross sectional area (Fig. 6C-F). In contrast, neither hypertension nor
microvascular remodeling occurred in arteries from DOX-treated Cyp4a12tg mice that received GPR75targeted shRNA (Fig. 6C-F). Taken together, these data strongly support the notion that activation of
GPR75 is a necessary step in 20-HETE-mediated hypertension, endothelial dysfunction, vascular smooth
muscle contractions and microvascular remodeling.
20-HETE-GPR75 pairing in vascular smooth muscle cells is associated with G
signaling.
q/11
and GIT1-mediated
20-HETE has been shown to stimulate vascular smooth muscle contraction via mechanisms that
include activation of PKC, MAPK and Src-type tyrosine kinases, all of which can phosphorylate and
inhibit the Ca2+-activated K+ channels (BKca), leading to depolarization, elevation in cytosolic [Ca2+], and
increased Ca2+ entry through the L-type Ca2+ channels.37 As seen in Figure 7, GPR75 is expressed in
cultured aortic vascular smooth muscle cells. In these cells, 20-HETE stimulated the dissociation of
Gq/11 from GPR75 (Fig. 7A), GPR75-GIT1 association (Fig. 7B), GIT1-PKC dissociation (Fig. 7C),
PKC-MaxiK association (Fig. 7D), c-Src-MaxiK association (Fig. 7E) and MaxiK tyrosine
phosphorylation (Fig. 7F). The phosphorylation of the MaxiKsubunit of the BKca channels leads to
inactivation of the channel and consequently to vasoconstriction.38
DOI: 10.1161/CIRCRESAHA.116.310525 8
DISCUSSION
Recent clinical studies and findings in animal models of genetically determined dysfunction
identified 20-HETE as a key lipid regulator of vascular, renal and cardiac functions. Numerous reports
documented causal relationships and polymorphic associations between levels of 20-HETE and its
biosynthetic enzymes with hypertension, vascular and renal injury, and cardiac hypertrophy.5 The
discovery of a receptor-ligand interaction between 20-HETE and the GPR75 orphan receptor, capable of
modulating some of the published effects of 20-HETE in the regulation of vascular function and blood
pressure, represents a major breakthrough in this area of research; it provides the molecular basis for the
signaling and pathophysiological functions mediated by 20-HETE and is the first identification of a
cytochrome P450-derived eicosanoid receptor.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
The presence of a specific cellular receptor/target for 20-HETE has been contemplated since the
first demonstration of its occurrence within the renal and cerebral microcirculation and its distinctive roles
in the regulation of the myogenic tone. The identification of a specific receptor, responsible for the
bioactive properties of 20-HETE, posed significant difficulties as 20-HETE is chemically and
metabolically labile and, as a lipid, it can cross membranes, and is rapidly esterified into phospholipids, or
effectively binds/sticks to proteins. To overcome some of these difficulties, we employed the 20-HETE
analog, 20-APheDa, and click chemistry methodology coupled with protein partner identification to
expose a multi-protein complex that is associated with 20-HETE binding in EC. The use of 20-APheDa
allowed us to take on a broader approach and obtain clues towards identifying receptor candidates. Clickchemistry compounds have been proven to be useful for labeling and used for the identification of multiprotein complexes and interactions.30 In these experiments, we aimed at potentially labeling several
proteins in close proximity to 20-APheDa in its bound and docked position. Several in-gel 20-APheDaprotein complexes were observed including a dominant band, which became the focus of our analysis.
This band revealed 20-APheDa’s effective labeling of several proteins and it was not until this
information was used for protein partner analysis that we were able to identify a candidate receptor
GPR75, a member of the rhodopsin Gq receptor family, as the putative GPCR. The ligand-receptor
pairing of 20-HETE with GPR75 was further demonstrated by competition binding studies in EC
membranes leading to Kd values that are well within the concentration range of 20-HETE biological
actions. Moreover, the finding that GPR75-deficient EC do not bind 20-HETE clearly support the
hypothesis that GPR75 is the receptor target for 20-HETE in these cells. In line with the fact that GPR75
is a Gq/11-coupled receptor are the demonstrations that 20-HETE increases IP-1 accumulation, an effect
not present in GPR75-deficient cells, and that 20-HETE rapidly dissociates Gq/11 from GPR75. All
together, these results indicate that 20-HETE binds to EC membranes, pairs with GPR75 and functionally
activates it.
In 1999, Tarttelin et al.,39 identified GPR75 as a novel human GPCR that maps to chromosome
2p16 and encodes a 540 amino acid protein. Initial findings showed GPR75 to be predominantly
expressed in cells surrounding retinal arterioles and in other areas of the brain.39, 40 Numerous databases
indicated a broad expression profile for Gpr5 in the majority of human tissues, and our qPCR assessment
in tissues from C57BL/6 corroborated this expression profile. Ignatov et al., reported that the chemokine
RANTES (CCL5) increased IP3 and intracellular Ca2+ in CHO or HEK cells overexpressing the mouse
GPR75 via a Gq protein-coupled PLC-mediated signal transduction.35 No direct binding studies were
documented in that study. A recent study by Liu et al. showed that CCL5 stimulates insulin secretion in
isolated islets via PLC-activated Ca2+ influx in a GPR75-dependent manner; however, they also did not
document direct binding or interaction between GPR75 and CCL5.41 Importantly, the pairing of CCL5
and GPR75 could not be repeated in a recent -arrestin assay and the International Union of Basic and
Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR)
still classifies GPR75 as an orphan receptor.42, 43 Notably, in our hands, CCL5 failed to dissociate G q/11
from GPR75 in EC. Moreover, CCL5 lacked the ability to transactivate EGFR and induce ACE mRNA,
DOI: 10.1161/CIRCRESAHA.116.310525 9
two bioactions of 20-HETE that are initiated with the activation of GPR75. Nevertheless, we cannot
exclude the possibility of interactions between CCL5 and 20-HETE at the level of GPR75 whereby CCL5
facilitates, amplifies or hinders the binding of 20-HETE.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
We proposed GPR75 as a 20-HETE receptor based on two key original findings. First, when
applied to EC, 20-HETE binds and activates the receptor at a concentration which elicits a specific
EGFR-MAPK-IKK-NFkB signaling pathway to induce ACE transcription, uncouple eNOS and stimulate
cytokine production.22, 24, 34, 44 This activation involves dissociation of Gq/11 from GPR75 and increased
GIT1-GPR75 association following by c-Src-mediated 20-HETE-dependent EGFR phosphorylation, the
first step of 20-HETE signaling in EC. Moreover, both GPR75 and GIT1 are required not only for 20HETE-mediated transactivation of EGFR but also for the downstream effect of 20-HETE to induce ACE
in EC (Fig. 8). The dissociation of Gq/11 from GPR75 following addition of 20-HETE, most likely,
initiated a PLC-IP3 mediated increases in [Ca2+], a common signaling pathway for Gq-coupled receptor.
Hence, the demonstration that 20-HETE increases IP-1 levels in a GPR75-dependent manner further
indicates a functional GPR75-20-HETE pairing. In smooth muscle cells, inhibition of the BKca channels
underlies 20-HETE-mediated vasoconstriction.45 Here we link GPR75-20-HETE pairing to stimulation of
Gq/11 dissociation and GIT1-mediated PKC- and cSrc-stimulated tyrosine phosphorylation of
MaxiKwhich inactivates the channel 38, 46 and alters BKca subunit trafficking 47 leading to cell
depolarization, elevation in cytosolic [Ca2+] and stimulation of the contractile apparatus. The role of HIC5 in the vascular endothelium is unclear. HIC-5 has been shown to interact with GIT1 48 and co-activate
the androgen receptor.49 The interaction between HIC-5 and the androgen receptor may account for the
strong relationship between androgens and 20-HETE. 50
The second key finding in this study is that the expression of GPR75 is required for the 20-HETE
pro-hypertensive activities. The conditional Cyp4a12tg mice display DOX-mediated hypertension along
with vascular dysfunction and remodeling in a 20-HETE-dependent manner.29, 33 20-HETE-mediated
vascular dysfunction and remodeling has been shown to be largely independent of blood pressure
elevation. 33, 36 We hypothesized that if GPR75 is the 20-HETE receptor, its disruption should prevent
hypertension in the DOX-treated Cyp4a12tg mice. Indeed, knockdown of GPR75 in DOX-treated
Cyp4a12tg mice prevented the 20-HETE-dependent hypertension along with marked reduction in
endothelial dysfunction, smooth muscle contractility, and vascular remodeling. These results clearly place
GPR75 as a novel target in the control of blood pressure and vascular function. Until recently, there were
no observed associations between GPR75 and hypertension. There is, however, a report that identified
GPR75 among other potential candidate genes in a locus on chromosome 2p which shows significant
linkage to anti-hypertensive responses in the British Genetics of Hypertension Study.51
As indicated above, GPR75 has been shown to express in a wide variety of tissues including the
brain, heart and kidney. All of these tissues participate in blood pressure control and have been shown to
have the capacity to produce 20-HETE. Accordingly, one may expect the presence of 20-HETE-GPR75
pairing in these tissues that may also contribute to hypertension. To this end, we found that GPR75 is also
expressed along the nephron, primarily on the luminal side of the proximal tubules (unpublished data)
where 20-HETE is presumably activating the NHE3 via angiotensin II-dependent mechanisms.52
Interestingly, salt-sensitive hypertension in mice expressing the human CYP4A11-20-HETE synthase was
attributed to enhanced Na retention resulting from 20-HETE- and Ang II-dependent NCC upregulation.53
Salt-sensitive hypertension was also shown in rats with depressed renal medullary 20-HETE synthesis,
which conditions Na retention in the ascending limb of the loop of Henle.54-57 Inasmuch as 20-HETE
contributes to pro-hypertensive mechanisms via vascular and tubular actions that foster vasoconstriction
and/or conservation of sodium, and to anti-hypertensive mechanisms via actions that facilitate Na
excretion, it is expected that the blood pressure effect of interventions that interfere with 20-HETE actions
is conditioned by both mechanisms. The discovery of 20-HETE-GPR75 pairing provides a unique
opportunity to confront the seemingly opposing actions of 20-HETE on blood pressure and explore the
DOI: 10.1161/CIRCRESAHA.116.310525 10
contribution of GPR75-20-HETE interactions in other tissues, including the central nervous system, to
hypertension.
In summary, we provide strong evidence for a 20-HETE-GPR75 pairing in the vascular
endothelium. We also present substantial data demonstrating that GPR75 is a critical component of 20HETE’s signaling and bioactions in vitro and in vivo, regulating vascular tone and function. This finding
opens the door for discovery and there is much to be explored including the role of this pairing in other
sites and organs such as the nervous, endocrine, and respiratory systems. It has the potential to be a game
changer in the field of CYP eicosanoids by providing novel targets for the development of new therapies
for myriad diseases/pathologies associated with 20-HETE (e.g., stroke, myocardial infarction) while
genetic studies of receptor variants could lead to a better understanding of some discrepancies between
20-HETE levels and reported pro- or anti-hypertensive properties.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
ACKNOWLEDGEMENT
We would like to thank Dr. Alberto Nasjletti for his thoughtful comments and constructive critiques in
planning the experiments and writing the manuscript. We also like to thank Dr. Houli Jiang in helping
with the generation of radiolabeled 20-HETE and Ms. Danielle Astarita in performing the
immunohistochemistry.
SOURCE OF FUNDING
This study was supported in part by National Institute of Health grants HL034300 (M.L. Schwartzman),
DK038226 (J.R. Falck and J.H. Capdevila); the Robert A. Welch Foundation (I-0011, J.R. Falck); the
National Heart, Lung, and Blood Institute Diversity Supplement Award HL34300-26A1S1 (V. Garcia),
Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (NIH Z01
ES025034 to DCZ); and the American Heart Association Undergraduate Summer Student Fellowship (B.
Shkolnik).
DISCLOSURES
None.
DOI: 10.1161/CIRCRESAHA.116.310525 11
REFERENCES
1.
2.
3.
4.
5.
6.
7.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Mancia G. Introduction to a compendium on hypertension. Circ Res. 2015;116:923-4.
Capdevila JH, Falck JR and Estabrook RW. Cytochrome P450 and the arachidonate cascade.
FASEB J. 1992;6:731-736.
Williams JM, Murphy S, Burke M and Roman RJ. 20-hydroxyeicosatetraeonic acid: a new target
for the treatment of hypertension. Journal of cardiovascular pharmacology. 2010;56:336-44.
Roman RJ. P-450 metabolites of arachidonic Acid in the control of cardiovascular function.
Physiological reviews. 2002;82:131-185.
Wu CC, Gupta T, Garcia V, Ding Y and Schwartzman ML. 20-HETE and blood pressure
regulation: clinical implications. Cardiol Rev. 2014;22:1-12.
Zordoky BN and El-Kadi AO. Effect of cytochrome P450 polymorphism on arachidonic acid
metabolism and their impact on cardiovascular diseases. Pharmacology & therapeutics.
2010;125:446-63.
Fava C, Montagnana M, Almgren P, Rosberg L, Lippi G, Hedblad B, Engstrom G, Berglund G,
Minuz P and Melander O. The V433M variant of the CYP4F2 is associated with ischemic stroke in
male Swedes beyond its effect on blood pressure. Hypertension. 2008;52:373-80.
Gainer JV, Bellamine A, Dawson EP, Womble KE, Grant SW, Wang Y, Cupples LA, Guo CY,
Demissie S, O'Donnell CJ, Brown NJ, Waterman MR and Capdevila JH. Functional variant of
CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension.
Circulation. 2005;111:63-9.
Fu Z, Nakayama T, Sato N, Izumi Y, Kasamaki Y, Shindo A, Ohta M, Soma M, Aoi N, Sato M,
Ozawa Y and Ma Y. Haplotype-based case-control study of CYP4A11 gene and myocardial
infarction. Hereditas. 2012;149:91-8.
Alexanian A, Miller B, Roman RJ and Sorokin A. 20-HETE-producing enzymes are up-regulated
in human cancers. Cancer genomics & proteomics. 2012;9:163-9.
Ward NC, Rivera J, Hodgson J, Puddey IB, Beilin LJ, Falck JR and Croft KD. Urinary 20hydroxyeicosatetraenoic acid is associated with endothelial dysfunction in humans. Circulation.
2004;110:438-443.
Ward NC, Puddey IB, Hodgson JM, Beilin LJ and Croft KD. Urinary 20-hydroxyeicosatetraenoic
acid excretion is associated with oxidative stress in hypertensive subjects. Free Radic Biol Med.
2005;38:1032-6.
Issan Y, Hochhauser E, Guo A, Gotlinger KH, Kornowski R, Leshem-Lev D, Lev E, Porat E, Snir
E, Thompson CI, Abraham NG and Laniado-Schwartzman M. Elevated level of pro-inflammatory
eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia. Prostaglandins Other
Lipid Mediat. 2013.
Tsai IJ, Croft KD, Mori TA, Falck JR, Beilin LJ, Puddey IB and Barden AE. 20-HETE and F2isoprostanes in the metabolic syndrome: the effect of weight reduction. Free Radic Biol Med.
2009;46:263-70.
Gervasini G, Luna E, Garcia-Cerrada M, Garcia-Pino G and Cubero JJ. Risk factors for posttransplant diabetes mellitus in renal transplant: role of genetic variability in the cyp450-mediated
arachidonic acid metabolism. Mol Cell Endocrinol. 2015.
Klawitter J, Klawitter J, McFann K, Pennington AT, Abebe KZ, Brosnahan G, Cadnapaphornchai
MA, Chonchol M, Gitomer B, Christians U and Schrier RW. Bioactive lipid mediators in
polycystic kidney disease. Journal of lipid research. 2013;55:1139-1149.
Dreisbach AW, Smith SV, Kyle PB, Ramaiah M, Amenuke M, Garrett MR, Lirette ST, Griswold
ME and Roman RJ. Urinary CYP eicosanoid excretion correlates with glomerular filtration in
African-Americans with chronic kidney disease. Prostaglandins Other Lipid Mediat. 2014;113115:45-51.
DOI: 10.1161/CIRCRESAHA.116.310525 12
18.
19.
20.
21.
22.
23.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Barden AE, Burke V, Mas E, Beilin LJ, Puddey IB, Watts GF, Irish AB and Mori TA. n-3 fatty
acids reduce plasma 20-hydroxyeicosatetraenoic acid and blood pressure in patients with chronic
kidney disease. Journal of hypertension. 2015.
Miyata N and Roman RJ. Role of 20-hydroxyeicosatetraenoic acid (20-HETE) in vascular system. J
Smooth Muscle Res. 2005;41:175-93.
Hoopes SL, Garcia V, Edin ML, Schwartzman ML and Zeldin DC. Vascular actions of 20-HETE.
Prostaglandins Other Lipid Mediat. 2015;120:9-16.
Cheng J, Edin ML, Hoopes SL, Li H, Bradbury JA, Graves JP, DeGraff LM, Lih FB, Garcia V,
Shaik JS, Tomer KB, Flake GP, Falck JR, Lee CR, Poloyac SM, Schwartzman ML and Zeldin DC.
Vascular characterization of mice with endothelial expression of cytochrome P450 4F2. Faseb J.
2014;28:2915-2931.
Cheng J, Garcia V, Ding Y, Wu CC, Thakar K, Falck JR, Ramu E and Schwartzman ML. Induction
of Angiotensin-Converting Enzyme and Activation of the Renin-Angiotensin System Contribute to
20-Hydroxyeicosatetraenoic Acid-Mediated Endothelial Dysfunction. Arterioscler Thromb Vasc
Biol. 2012;32:1917-1924.
Cheng J, Ou JS, Singh H, Falck JR, Narsimhaswamy D, Pritchard KA, Jr. and Schwartzman ML.
20-Hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling. Am J
Physiol Heart Circ Physiol. 2008;294:H1018-26.
Garcia V, Shkolnik B, Milhau L, Falck JR and Schwartzman ML. 20-HETE Activates the
Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-kappaB Translocation and
Promoter Binding. J Pharmacol Exp Ther. 2016;356:525-33.
Mehta PK and Griendling KK. Angiotensin II cell signaling: physiological and pathological effects
in the cardiovascular system. American journal of physiology. 2007;292:C82-97.
Okada H. A look at transactivation of the EGF receptor by angiotensin II. J Am Soc Nephrol.
2012;23:183-5.
Akbulut T, Regner KR, Roman RJ, Avner ED, Falck JR and Park F. 20-HETE activates the
Raf/MEK/ERK pathway in renal epithelial cells through an EGFR- and c-Src-dependent
mechanism. Am J Physiol Renal Physiol. 2009;297:F662-70.
Alonso-Galicia M, Falck JR, Reddy KM and Roman RJ. 20-HETE agonists and antagonists in the
renal circulation. Am J Physiol. 1999;277:F790-6.
Wu CC, Mei S, Cheng J, Ding Y, Weidenhammer A, Garcia V, Zhang F, Gotlinger K, Manthati
VL, Falck JR, Capdevila JH and Schwartzman ML. Androgen-sensitive hypertension associates
with upregulated vascular CYP4A12-20-HETE synthase. J Am Soc Nephrol. 2013;24:1288-96.
Hulce JJ, Cognetta AB, Niphakis MJ, Tully SE and Cravatt BF. Proteome-wide mapping of
cholesterol-interacting proteins in mammalian cells. Nature methods. 2013;10:259-64.
Kim-Kaneyama JR, Lei XF, Arita S, Miyauchi A, Miyazaki T and Miyazaki A. Hydrogen
peroxide-inducible clone 5 (Hic-5) as a potential therapeutic target for vascular and other disorders.
J Atheroscler Thromb. 2012;19:601-7.
Premont RT, Claing A, Vitale N, Freeman JL, Pitcher JA, Patton WA, Moss J, Vaughan M and
Lefkowitz RJ. beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinaseassociated ADP ribosylation factor GTPase-activating protein. Proc Natl Acad Sci U S A.
1998;95:14082-7.
Garcia V, Joseph G, Shkolnik B, Ding Y, Zhang FF, Gotlinger K, Falck JR, Dakarapu R, Capdevila
JH, Bernstein KE and Schwartzman ML. Angiotensin II receptor blockade or deletion of vascular
endothelial ACE does not prevent vascular dysfunction and remodeling in 20-HETE-dependent
hypertension. Am J Physiol Regul Integr Comp Physiol. 2015;309:R71-8.
Ishizuka T, Cheng J, Singh H, Vitto MD, Manthati VL, Falck JR and Laniado-Schwartzman M. 20Hydroxyeicosatetraenoic acid stimulates nuclear factor-kappaB activation and the production of
inflammatory cytokines in human endothelial cells. J Pharmacol Exp Ther. 2008;324:103-10.
DOI: 10.1161/CIRCRESAHA.116.310525 13
35.
36.
37.
38.
39.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Ignatov A, Robert J, Gregory-Evans C and Schaller HC. RANTES stimulates Ca2+ mobilization
and inositol trisphosphate (IP3) formation in cells transfected with G protein-coupled receptor 75.
British journal of pharmacology. 2006;149:490-7.
Ding Y, Wu CC, Garcia V, Dimitrova I, Weidenhammer A, Joseph G, Zhang F, Manthati VL,
Falck JR, Capdevila JH and Schwartzman ML. 20-HETE induces remodeling of renal resistance
arteries independent of blood pressure elevation in hypertension. Am J Physiol Renal Physiol.
2013;305:F753-63.
Garcia V and Schwartzman ML. Recent developments on the vascular effects of 20hydroxyeicosatetraenoic acid. Curr Opin Nephrol Hypertens. 2016.
Alioua A, Mahajan A, Nishimaru K, Zarei MM, Stefani E and Toro L. Coupling of c-Src to large
conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced
vasoconstriction. Proc Natl Acad Sci U S A. 2002;99:14560-5.
Tarttelin EE, Kirschner LS, Bellingham J, Baffi J, Taymans SE, Gregory-Evans K, Csaky K,
Stratakis CA and Gregory-Evans CY. Cloning and characterization of a novel orphan G-proteincoupled receptor localized to human chromosome 2p16. Biochemical and biophysical research
communications. 1999;260:174-80.
Sauer CG, White K, Stohr H, Grimm T, Hutchinson A, Bernstein PS, Lewis RA, Simonelli F,
Pauleikhoff D, Allikmets R and Weber BH. Evaluation of the G protein coupled receptor-75
(GPR75) in age related macular degeneration. The British journal of ophthalmology. 2001;85:96975.
Liu B, Hassan Z, Amisten S, King AJ, Bowe JE, Huang GC, Jones PM and Persaud SJ. The novel
chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to
stimulation of insulin secretion and improved glucose homeostasis. Diabetologia. 2013;56:2467-76.
Southern C, Cook JM, Neetoo-Isseljee Z, Taylor DL, Kettleborough CA, Merritt A, Bassoni DL,
Raab WJ, Quinn E, Wehrman TS, Davenport AP, Brown AJ, Green A, Wigglesworth MJ and Rees
S. Screening beta-arrestin recruitment for the identification of natural ligands for orphan G-proteincoupled receptors. J Biomol Screen. 2013;18:599-609.
Davenport AP, Alexander SP, Sharman JL, Pawson AJ, Benson HE, Monaghan AE, Liew WC,
Mpamhanga CP, Bonner TI, Neubig RR, Pin JP, Spedding M and Harmar AJ. International Union
of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations
for new pairings with cognate ligands. Pharmacological reviews. 2013;65:967-86.
Cheng J, Wu CC, Gotlinger KH, Zhang F, Falck JR, Narsimhaswamy D and Schwartzman ML. 20hydroxy-5,8,11,14-eicosatetraenoic acid mediates endothelial dysfunction via IkappaB kinasedependent endothelial nitric-oxide synthase uncoupling. J Pharmacol Exp Ther. 2010;332:57-65.
Zou AP, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR and Roman RJ. 20-HETE
is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles.
AmJPhysiol. 1996;270:R228-R237.
Obara K, Koide M and Nakayama K. 20-Hydroxyeicosatetraenoic acid potentiates stretch-induced
contraction of canine basilar artery via PKC alpha-mediated inhibition of KCa channel. British
journal of pharmacology. 2002;137:1362-70.
Kyle BD and Braun AP. The regulation of BK channel activity by pre- and post-translational
modifications. Frontiers in physiology. 2014;5:316.
Nishiya N, Shirai T, Suzuki W and Nose K. Hic-5 interacts with GIT1 with a different binding
mode from paxillin. Journal of biochemistry. 2002;132:279-89.
Heitzer MD and DeFranco DB. Hic-5/ARA55: a prostate stroma-specific AR coactivator. Steroids.
2007;72:218-20.
Wu CC, Cheng J, Zhang FF, Gotlinger KH, Kelkar M, Zhang Y, Jat JL, Falck JR and Schwartzman
ML. Androgen-Dependent Hypertension Is Mediated by 20-Hydroxy-5,8,11,14-Eicosatetraenoic
Acid-Induced Vascular Dysfunction: Role of Inhibitor of {kappa}B Kinase. Hypertension.
2011;57:788-94.
DOI: 10.1161/CIRCRESAHA.116.310525 14
51.
52.
53.
54.
55.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
56.
57.
Padmanabhan S, Wallace C, Munroe PB, Dobson R, Brown M, Samani N, Clayton D, Farrall M,
Webster J, Lathrop M, Caulfield M, Dominiczak AF and Connell JM. Chromosome 2p shows
significant linkage to antihypertensive response in the British Genetics of Hypertension Study.
Hypertension. 2006;47:603-8.
Quigley R, Chakravarty S, Zhao X, Imig JD and Capdevila JH. Increased renal proximal
convoluted tubule transport contributes to hypertension in Cyp4a14 knockout mice. Nephron
Physiol. 2009;113:p23-8.
Savas U, Wei S, Hsu MH, Falck JR, Guengerich FP, Capdevila JH and Johnson EF. 20Hydroxyeicosatetraenoic Acid (HETE)-dependent Hypertension in Human Cytochrome P450
(CYP) 4A11 Transgenic Mice: NORMALIZATION OF BLOOD PRESSURE BY SODIUM
RESTRICTION, HYDROCHLOROTHIAZIDE, OR BLOCKADE OF THE TYPE 1
ANGIOTENSIN II RECEPTOR. J Biol Chem. 2016;291:16904-19.
Roman RJ, Ma YH, Frohlich B and Markham B. Clofibrate prevents the development of
hypertension in Dahl salt-sensitive rats. Hypertension. 1993;21:985-8.
Stec DE, Mattson DL and Roman RJ. Inhibition of renal outer medullary 20-HETE production
produces hypertension in Lewis rats. Hypertension. 1997;29:315-9.
Roman RJ, Alonso-Galicia M and Wilson TW. Renal P450 metabolites of arachidonic acid and the
development of hypertension in Dahl salt-sensitive rats. Am J Hypertens. 1997;10:63s-67s.
Ito O and Roman RJ. Role of 20-HETE in elevating chloride transport in the thick ascending limb
of Dahl SS/Jr rats. Hypertension. 1999;33:419-23.
DOI: 10.1161/CIRCRESAHA.116.310525 15
FIGURE LEGENDS
Figure 1: Isolation of a 20-HETE-associated membrane protein complex by the click-in chemistry.
A)
Structures
of
the
pharmacological
probe
20-azido-N-4-(3-(4benzoylphenylpropanamido)phenylsulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide (20-APheDa)
and 20-HETE. B) Cumulative concentration-response curve to phenylephrine in renal interlobar arteries
from C57BL/6 mice preincubated with the 20-HETE biosynthesis inhibitor DDMS (30 µmol/L) and
cumulative concentration responses to phenylephrine (10-3 to 102 µmol/L) was constructed in the
presence and absence of 20-HETE (10 µmol/L) or 20-HETE (10 µmol/L) +20-APheDa (10 µmol/L)
(*p<0.05 vs DDMS; 3p<0.05 vs DDMS+20-HETE; mean±SEM, n=6). C) A representative (n=8) In-gel
image band composition of 20-APheDa (10 nmol/L) bound to human microvascular endothelial cell
membrane proteins (ECm, 20µg). D-E) Binding of 20-APheDa (0.1 nmol/L) to ECm (10 µg) is competed
for by 20-HETE (100 μmol/L) but not by 12(S)-HETE (100 μmol/L) (representative image, n=5-6). The
line between the marker lane (M) and the ECm lane in panels C-E reflects the fact that although the lanes
are part of the same gel they are on a separate infrared channel.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Figure 2: 20-HETE binds to GPR75. A) Displacement of radiolabeled [3H8] 20-HETE in membranes by
unlabeled 20-HETE and 12(S)-HETE. B) Assessment of Kd. C) GPR75 protein levels in EC treated with
control and GPR75-specific siRNA for 36 h. D) Displacement of radiolabeled [3H8] 20-HETE (6.67
nmol/L) by increasing concentrations of unlabeled 20-HETE in membranes from EC treated with control
and GPR75-specific siRNA for 36 h. E) Assessment of Kd from displacement bindings in 3D. F)
Displacement of [3H8] 20-HETE by 20-6,15-HEDGE (1 nmol/L). G-H) Effect of 20-HETE (10 nmol/L)
on IP-1 accumulation in EC treated with control and GPR75-specific siRNAs. All results are mean±SEM
(n=4-6, *p<0.05 vs. vehicle, #p<0.05 vs. 20-HETE)
Figure 3: Expression of GPR75 in the vascular endothelium and effect of 20-HETE on GPR75
association with GIT1, HIC-5 and Gq/11 in EC. A) A representative image of GPR75 (red) and CD31
(green) immunofluorescence of kidney sections from C57BL/6 mice (20X; scale bar, 10µm). B) A
representative Western blot of GPR75 immunoprecipitate showing association with Gq/11, GIT1 and
HIC-5 in EC. 20-HETE alters GPR75 association with (C) Gq/11, (D) GIT1 and (E) HIC-5. F) Effect of
20-6,15-HEDGE on 20-HETE-stimulated GPR75-GIT1 association. EC were incubated with 20-HETE
(1 or 10 nmol/L) with and without 20-6,15-HEDGE (10 nmol/L) for 5 min and GPR75 was
immunoprecipitated and immunoblot for the indicated proteins (mean±SEM, n=4, *p<0.05 vs Vehicle,
#
p<0.05 vs 20-HETE).
Figure 4. GPR75 and GIT1 are required for 20-HETE signaling in EC (A-C). EC were transfected
with control siRNA, GPR75- or GIT1-specific siRNA for 36 h. Cells were then incubated in a serum free
media for 12 h prior to addition of 20-HETE (10 nmol/L) for 5 min. A) Representative western blot
showing GPR75 and B) GIT1 knockdown. C) Effect of GPR75 and GIT1 knockdown on 20-HETEstimulated EGFR phosphorylation (immunoblots are from separate experiments noted by dividing line;
mean±SEM, n=4, *p<0.05 vs. Vehicle). 20-HETE stimulation of EGFR phosphorylation is mediated
by GPR75-GIT1-cSrc activation in EC (D-F). The effects of 20-HETE (10 nmol/L) treatment (5 min)
on: D) phosphorylated EGFR, E) c-Src bound to GIT and F) c-Src bound to EGFR, (mean±SEM, n=4,
*p<0.05 vs Vehicle). G) GPR75 knockdown prevents 20-HETE-mediated ACE induction in EC.
Endothelial ACE mRNA expression from control and GPR75 siRNA treated cells in the presence and
absence of 20-HETE (10 nmol/L) for 2 h (mean±SEM, n=3, *p<0.05 vs Vehicle, #p<0.05 vs 20-HETE).
DOI: 10.1161/CIRCRESAHA.116.310525 16
Figure 5: GPR75 Knockdown Prevents 20-HETE-Dependent Hypertension and ACE induction in
Cyp4a12tg Mice. A) Systolic blood pressure monitoring in Cyp4a12tg mice receiving water, control
shRNA+DOX and GPR75 shRNA+DOX (n=4, *p<0.05 vs water) for 14 days. B) Systolic blood pressure
of Cyp4a12tg mice at day 14 of treatment with water, DOX, control shRNA, control shRNA+DOX or
GPR75 shRNA+DOX. C) Vascular GPR75 expression in PGMVs from control and GPR75 shRNAtreated Cyp4a12tg mice at day 14. D) Vascular ACE expression in preglomerular arteries from
Cyp4a12tg mice receiving water, DOX, control shRNA, control shRNA+DOX and GPR75 shRNA+DOX
for 14 days. Blood pressure was measured by the tail cuff method. Results are mean±SEM, n=4, *p<0.05
vs water or control shRNA+DOX.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Figure 6: GPR75 knockdown prevent 20-HETE-dependent hypertension, vascular dysfunction and
vascular remodeling (A-F). A) Cumulative concentration-response curve to acetylcholine in renal
interlobar arteries from Cyp4a12tg mice treated with water, Control shRNA, DOX, control
shRNA+DOX, and GPR75 shRNA+DOX for 14 days (meaan±SEM, n=4, *p<0.05 vs water, #p<0.05 vs
control shRNA+DOX). B) Cumulative concentration-response curve to phenylephrine in renal interlobar
arteries from water, control and GPR75 shRNA treated mice receiving DOX after 12 days. EC50 and
Rmax are given in the upper inset (n=6, *p<0.05 vs water). C) Systolic blood pressure measurements (tail
cuff) in control and GPR75 shRNA-treated Cyp4a12tg mice receiving DOX for 35 days (n=3, *p<0.05 vs
water, #p<0.05 vs control shRNA+DOX). Vascular remodeling measured by pressure myography as D)
media thickness, E) media-to-lumen ratio and F) cross sectional area in renal interlobar arteries from mice
treated with control and GPR75 shRNA after receiving DOX for 35 days as previously described 39 (n=4,
*p<0.05 vs water, #p<0.05 vs control shRNA+DOX).
Figure 7: GPR75-20-HETE pairing in vascular smooth muscle is linked to inhibition of MaxiK
Cultured aortic vascular smooth muscle cells were treated with 20-HETE (10 nmol/L) for 5 min.
Immunoprecipitation followed by immunoblotting and densitometry analysis showed increases in: A)
Gq/11 dissociation from GPR75, B) GPR75-GIT1 association, C) GIT1-PKC dissociation, D) PKCMaxiK association, E) cSrc-MaxiK
association and F) MaxiK tyrosine phosphorylation
(mean±SEM, n=4, *p<0.05 vs Vehicle).
Figure 8: Proposed 20-HETE-GPR75-mediated signaling in endothelial cells. 20-HETE-GPR75
pairing stimulates the dissociation of G q/11 and the association of GIT1 to the receptor. The later
facilitates c-Src-mediated EGFR transactivation. The 20-HETE-GPR75-mediated activation of EGFR
results in the stimulation of downstream cascades that regulate vascular ACE expression and decreases in
NO bioavailability.
DOI: 10.1161/CIRCRESAHA.116.310525 17
NOVELTY AND SIGNIFICANCE
What Is Known?

20-hydroxyeicosatetraenoic acid (20-HETE), a bioactive lipid autacoid, plays a role in the
pathogenesis of hypertension, stroke, myocardial infarction, renal failure and diabetes.

The actions of 20-HETE appear to involve interaction with a specific receptor/target molecule,
yet, the cellular presence of a specific receptor for 20-HETE has not been demonstrated.
What New Information Does This Article Contribute?
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017

This study identifies GPR75, an orphan G-protein-coupled receptor (GPCR), as the putative 20HETE-receptor. This is the first time a GPCR has been identified for an eicosanoid of this class.

The experimental approach to identifying a specific receptor target for 20-HETE included the use
of a click chemistry crosslinking analog, proteomics, protein partner analysis, receptor binding
assays, gene silencing and functional assays

20-HETE binding to GPR75 activates distinct signaling cascades in endothelial and vascular
smooth muscle cells that culminate in the activation vascular ACE expression, endothelial
dysfunction, contractility, remodeling and hypertension.

The discovery of 20-HETE-GPR75 pairing opens the door for future research with regards to how
this lipid-receptor interaction is involved in a variety of pathologies known to be closely mediated
and associated with 20-HETE.
The high prevalence and the significant contribution of hypertension to cardiovascular and renal disease
comprise its position as the top contributor to the burden of disease worldwide. However, with few
exceptions, the molecular basis underlying the pathogenesis of hypertension remains to be defined.
Moreover, despite the large number of available treatment options, a significant portion of the
hypertensive population has uncontrolled blood pressure. The plethora of available anti-hypertensive
drugs, while lowering blood pressure, do not always correct the organ damage associated with
hypertension. 20-HETE has been identified as a significant contributing factor to the pathophysiology of
hypertension and its cardiovascular consequences. In this study, we identified a cell surface receptor, the
orphan GPCR (Gq) GPR75, to which 20-HETE binds to and initiates a cell-signaling cascade leading to
endothelial cell dysfunction and vascular smooth muscle contractility. This study further show that
knockdown of GPR75 prevents 20-HETE-mediated vascular remodeling and hypertension. The
identification of 20-HETE-GPR75 pairing opens the door for discovery and there is much to be explored
including the role of this interaction in a variety of diseases/pathologies associated with 20-HETE (e.g.,
stroke, myocardial infarction).
DOI: 10.1161/CIRCRESAHA.116.310525 18
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
FIGURE 1
O
O
A
benzophenone
phot o cross li nk er
O
S
NH
B
O
n
OH
cli ck sit e
Rmax
(mN/mm)
DDMS
6 2.43㼼0.47
DDMS+20-HETE 6 0.56㼼0.16*
DDMS+20-HETE 6 3.06㼼1.06#
+20-ApheDa
N
H
N3
EC50
(μmol/l)
O
20-APheDa
20-azido-N-((4-(3-(4-benzoylphenyl)propanamido)phenyl)sulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide
1.47㼼0.05
1.30㼼0.12
1.45㼼0.07
COOH
OH
20-HETE
20-hydroxyeicosatetraenoic acid
C
kDa
M
ECm
170
130
95
72
55
D
E
kDa
43
55
34
43
26
34
M
ECm
kDa
M
ECm
ECm
55
43
17
ECm
+
20-HETE
34
-
+
12(S)-HETE
B
A
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
0.10
FIGURE 2
1/ȴ CPM
y = 0.0008x + 0.003
R² = 0.9991
20-HETE
0.05
Kd=3.75nM
0.00
0
50
100
1/[20-HETE nM]
GPR75
- 55
ȕ-Actin
- 43
E
0.10
y = 0.0006x + 0.0044
R² = 0.9989
1/ȴ CPM
1.5
1
Control siRNA
0.05
Kd=7.33nM
0.5
*
0.00
0
0
G
600
400
200
*
0
Vehicle
50
100
1/[20-HETE nM]
GPR75
SiRNA
20-6,15-HEDGE
H
*
1800
1500
1200
900
600
300
0
Vehicle
20-HETE
IP-1 (Fold Change)
Control
SiRNA
F
Counts per Minutes (CPM)
D
IP-1 Accumulation [nM]
GPR75/ȕ-Actin
(Relative to Vehicle)
C
8
*
Vehicle
20-HETE
6
4
#
2
0
Control
SiRNA
GPR75
SiRNA
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
B
C
20-HETE
GPR75
CD31
FIGURE 3
IP: GPR75
GPR75
GIT1
20-HETE (nM)
Vehicle
HIC-5 GĮq/11
GĮq/11
IP: GPR75
- 95
WB: GIT1
WB: HIC-5
- 43
GDq/11/GPR75
WB: GĮq/11
- 55
WB: GPR-75
E
1.2
0.8
Vehicle 20-HETE
1
0
Vehicle
20-HETE
- 43
- 55
GPR75
HIC-5/GPR75
2
Vehicle
1nM
10nM
0.0
*
0.5
0.0
Vehicle
20-HETE
20-6,
20-HETE
20-6,15-HEDGE 15-HEDGE
- 95
GPR75
- 55
1.5
1.0
20-HETE
GIT1
(Relative to Vehicle)
HIC-5
3
*
*
0.4
IP: GPR75
IP: GPR75
GIT1/GPR75
GPR75
- 55
(Relative to Vehicle)
- 95
GIT1/GPR75
GIT1
*
F
(Relative to Vehicle)
Vehicle 20-HETE
- 43
- 55
Vehicle
IP: GPR75
10
EC
- 43
D
1
GPR75
(Relative to Vehicle)
A
3
2
*
#
1
0
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
A
Control siRNA
GPR75 siRNA
Vehicle 20-HETE Vehicle 20-HETE
Control siRNA
B
GIT1 siRNA
Vehicle 20-HETE Vehicle
FIGURE 4
20-HETE
GPR75
- 55
GIT1
-95
Actin
- 43
Actin
- 43
C
IP: EGFR
Control siRNA
D
GPR75 siRNA
IP: EGFR
GIT1 siRNA
Vehicle 20-HETE Vehicle 20-HETE Vehicle 20-HETE
P-Tyr
P-Tyr
EGFR
- 170
EGFR
- 170
3
P-Tyr/EGFR
P-Tyr/EGFR
3
*
2
1
0
GIT1 siRNA
G
F IP: EGFR
Vehicle 20-HETE
- 72
Vehicle 20-HETE
- 55
c-Src
GIT1
- 95
EGFR
2
1
*
0
Vehicle
20-HETE
c-Src/EGFR
c-Src
- 72
- 55
- 170
3
*
2
1
- 170
*
2
1
Vehicle
ACE mRNA
IP: GIT1
c-Src/GIT1
E
GPR75 siRNA
- 170
0
Vehicle 20-HETE Vehicle 20-HETE Vehicle 20-HETE
Control siRNA
Vehicle 20-HETE
20-HETE
5
4
*
3
2
#
1
0
0
Vehicle
20-HETE
Vehicle 20-HETE Vehicle 20-HETE
GPR75 siRNA
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
B
Water
Control ShRNA+DOX
GPR75 shRNA+DOX
160
150
*
*
140
130
*
shRNA
Injection
120
110
100
90
-2
0
2
5
8
12
Days on DOX
C
D
- 43
ȕ-Actin
0.0
Control
shRNA
GPR75
shRNA
ACE/ȕ-Actin
*
(Relative to Vehicle)
ȕ-Actin
GPR75/ȕ-Actin
(Relative to Vehicle)
GPR75
1.5
*
*
130
120
110
100
90
80
Water
DOX
Control
shRNA
Control
shRNA +
DOX
GPR75
shRNA +
DOX
- 200
ACE
0.5
140
14
- 55
1.0
FIGURE 5
Systolic Blood Pressure (mmHg)
Systolic Blood Pressure (mmHg)
A
- 43
5
*
*
4
3
2
1
0
Water
DOX
Control
shRNA
Control
shRNA +
DOX
GPR75
shRNA +
DOX
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
5
10
50
100
% Relaxation
-20
-40
*
-60
*
-80
#
-100
Water
Control shRNA
DOX
Control shRNA+DOX
GPR75 shRNA+DOX
8
7
6 0.75㼼0.11
6 0.40㼼0.08*
6 0.86㼼0.24#
4.89㼼0.51
6.17㼼0.39*
4.54㼼0.46
6
5
4
3
2
Vehicle control
Control shRNA+DOX
GPR75 shRNA+DOX
1
0
0.001 0.01
0.1
1
10
*
Media:Lumen
20
15
10
*
0.3
0.2
0.1
0.0
0
GPR75
shRNA+
DOX
*
140
130
120
110
100
90
80
Control
shRNA
Control
GPR75
shRNA + shRNA +
DOX
DOX
25
5
Control
shRNA +
DOX
100
0.4
25
Control
shRNA
FIGURE 6
F
E
30
C
Rmax
(mN/mm)
Phenylephrine (μM )
D
Media Thickness (μm)
n
EC50
(μmol/l)
20
(μm2 x103)
1
ǻ,VRPHWULF7HQVLRQP1PP
0
0.5
Systolic Blood Pressure (mmHg)
B
ACH (ȝmol/l)
0.01 0.05 0.1
Cross Sectional Area
A
*
15
10
5
0
Control Control GPR75
shRNA shRNA + shRNA+
DOX
DOX
Control Control GPR75
shRNA shRNA + shRNA+
DOX
DOX
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
20-HETE
- 43
GPR75
- 55
2
1
*
1
0
Vehicle
20-HETE
20-HETE
3
Vehicle 20-HETE
- 95
PKCD
- 95
- 55
GIT1
- 95
2
*
2
1
0
Vehicle
20-HETE
PKCD
- 43
MaxiKȕ
PKCĮ/MaxiKȕ
- 95
- 34
3
*
2
1
0
Vehicle
20-HETE
20-HETE
F
IP: MaxiKȕ
Vehicle
Vehicle
20-HETE
c-SRC
- 55
- 43
MaxiKȕ
c-SRC/MaxiKȕ
Vehicle
*
Vehicle
IP: MaxiKȕ
IP: MaxiKȕ
1
0
20-HETE
E
D
FIGURE 7
IP: GIT1
GIT1
GPR75
GIT1/GPR75
GDq/11/GPR75
IP: GPR75
Vehicle
C
PKCĮ/GIT1
IP: GPR75
Vehicle
GDq/11
B
20-HETE
P-Tyr
3
*
2
1
0
Vehicle
20-HETE
- 43
- 34
- 43
MaxiKȕ
- 34
- 34
P-Tyr/MaxiKȕ
A
3
*
2
1
0
Vehicle
20-HETE
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
FIGURE 8
Step 1: 20-HETE Binds and Activates GPR75:
Releases GĮq/11 and HIC-5
GIT1-GPR75 Binding Increases
20-HETE
GPR75
HIC-5
EGFR
GIT1
c-Src
GĮq/11
Step 2: Released GĮq/11 Activates Calcium Release
Bound c-Src is Released from GIT1
EGFR
GPR75
GIT1
c-Src
HIC-5
PLC
IP3
DAG
PKC
c-Src
GĮq/11
Ca2+
Step 3: Released c-Src Binds to and Phosphorylates EGFR
EGFR
GPR75
HIC-5
GIT1
P
c-Src
ACE
P
GĮq/11
MAPK
IKK Į/ȕ
NF-țB
HSP90
IțB
eNOS
NO
O2-
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
20-HETE Signals Through G Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular
Function and Trigger Hypertension
Victor Garcia, Ankit Gilani, Brian Shkolnik, Varunkumar Pandey, Frank F Zhang, Rambabu
Dakarapu, Shyam K Gandham, N R Reddy, Joan P Graves, Artiom Gruzdev, Darryl C Zeldin, Jorge
H Capdevila, John R Falck and Michal L Schwartzman
Circ Res. published online March 21, 2017;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2017 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2017/03/23/CIRCRESAHA.116.310525
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2017/03/21/CIRCRESAHA.116.310525.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this process is
available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
1
SUPPLEMENTAL MATERIAL
20- HETE signals through G protein-coupled receptor GPR75 (Gq) to affect vascular
function and trigger hypertension
Victor Garcia, PhDa, Ankit Gilani, MSa, Brian Shkolnik, BSa, Varunkumar Pandey, PhDa,
Frank Fan Zhang, MD,PhDa, Rambabu Dakarapu, PhDb, Shyam K. Gandham, PhDb,
N. Rami Reddy, PhDb, Joan P. Graves, MSc, Artiom Gruzdev, PhDc, Darryl C. Zeldin, MDc,
Jorge H. Capdevila, PhDd, John R. Falck, PhDb and Michal Laniado Schwartzman, PhDa
a
Department of Pharmacology, New York Medical College School of Medicine, Valhalla, NY
10595
b
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX
75390
c
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
d
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37240
2
Material and Methods
Synthesis
of
Photoactivated
Cross-Linker
20-azido-N-((4-(3-(4benzoylphenyl)propanamido)-phenyl)sulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide
(20-APheDa).
Nuclear magnetic resonance (NMR) spectra were recorded on Varian 300, 400 or 500
spectrometers at operating frequencies of 300/400/500 MHz (1H) or 75/100/125 MHz (13C) in
CDCl3 with TMS as internal standard, unless otherwise stated. 1H NMR data are reported as
follows: chemical shift, 1 multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q =
quartet, app q = apparent quartet, qn = quintet, app qn = apparent quintet, m = multiplet) and
coupling constant (Hz). High resolution mass spectra (HRMS) were obtained at UT-Arlington
using a Shimadzu IT-TOF mass spectrometer or at the Medical College of Wisconsin by Prof.
Michael Thomas. Infrared (IR) spectra were obtained using a Perkin Elmer Spectrum 1000
Fourier transform spectrometer. Melting points were measured using an OptiMelt from Stanford
Research Systems and are uncorrected. Analytical thin layer chromatography (TLC) used EMD
Chemicals TLC silica gel 60 F254 plates (0.040-0.063 mm) with visualization by UV light and/or
KMnO4 or phosphomolybdic acid (PMA) solution followed by heating. All oxygen and/or
moisture sensitive reactions were performed under an argon atmosphere using oven-dried
glassware and anhydrous solvents. Extracts were dried over anhydrous Na2SO4 and filtered
prior to removal of all volatiles under reduced pressure. Chromatographic purifications utilized
preparative TLC or flash chromatography using pre-packed SiO2 columns on a CombiFlash
Rf200 chromatograph (Teledyne Isco). Unless otherwise noted, yields refer to isolated and
purified material with spectral data consistent with assigned structures or, if known, were in
agreement with published data. Reagents were purchased at the highest commercial quality
available and used without further purification, unless otherwise noted. Anhydrous solvents
were dried using a Glass Contours Solvent System by passage through columns of activated
packing material under argon immediately prior to use.
O
O O
S
H2N
EDCI HCl
HO
NH2
O O
S
H2N
O
N
H
O
O
Solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl) (255 mg, 1.33
mmol) was added in portions to a stirring, rt solution 3-(4-benzoylphenyl)propanoic acid (200
mg, 0.786 mmol), 2 commercial p-sulfanilamide (135 mg, 0.786 mmol), and
hydroxybenzotriazole (HOBT) (116 mg, 0.865 mmol) in anhydrous DMF (5 mL). After 18 h, the
reaction mixture was diluted with water (20 mL). The resultant precipitate was collected by
filtration, washed with water (30 mL) and dried in vacuo at 30 °C to give 3-(4-benzoylphenyl)-N(4-sulfamoylphenyl)propanamide (224 mg, 70%) as a white powder. TLC: 80% EtOAc/hexanes,
Rf ≈ 0.5.
1
H NMR (400 MHz, DMSO) δ 10.29 (s, 1H), 7.82–7.64 (m, 9H), 7.57 (d, J = 8.0, 2H), 7.45 (d, J
= 8.0, 2H), 7.24 (s, 2H), 3.03 (t, J = 7.6, 2H), 2.75 (d, J = 7.6, 2H).
COOMe
N3
OH
TBDMS Cl
Imidazole
COOMe
N3
OTBDMS
Imidazole (59 mg, 0.86 mmol), TBDMS-Cl (52 mg, 0.34 mmol), and DMAP (5 mg) were added
to a stirring, 0 °C solution of methyl 20-azido-19(S)-hydroxyeicosa-5(Z),14(Z)-dienoatea (110 mg,
0.28 mmol) in dry CH2Cl2 (15 mL). After stirring at room temperature for 48 h, the reaction
mixture was quenched with water (5 mL) and extracted with CH2Cl2 (3 × 6 mL). The combined
3
organic extracts were washed with brine (10 mL), dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by SiO2 column chromatography using
hexanes/EtOAc (94:6) to give methyl 20-azido-19(S)-(tert-butyldimethylsilyloxy)eicosa5(Z),14(Z)-dienoate (134 mg, 94%) as a colorless oil. TLC: 20% EtOAc/hexane, Rf ≈ 0.6.1H
NMR (CDCl3, 400 MHz) δ 5.45–5.24 (m, 4H), 3.76–3.65 (m, 1H), 3.65 (s, 3H), 3.23 (dd, J =
12.4, 4.1 Hz, 1H), 3.12 (dd, J = 12.5, 5.9 Hz, 1H), 2.30 (t, J = 7.5 Hz, 2H), 2.11–1.92 (m, 8H),
1.74–1.63 (m, 2H), 1.58–1.43 (m, 2H), 1.43–1.22 (m, 12H), 0.89 (s, 9H), 0.09 (s, 3H), 0.07 (s,
3H); 13C NMR (CDCl3, 101 MHz) δ 174.07, 131.09, 130.50, 129.05, 128.31, 71.69, 56.62, 51.42,
34.65, 33.42, 29.70, 29.67, 29.43, 29.26, 27.25, 27.19, 27.15, 26.51, 25.77, 25.21, 24.86, 17.99,
-4.64, -4.67.
COOMe
COOH
LiOH
N3
OTBDMS
N3
OTBDMS
A solution of LiOH (0.8 mL of 1 M aq. soln, 0.78 mmol) and methyl 20-azido-19(S)-(tertbutyldimethylsilyloxy)eicosa-5(Z),14(Z)-dienoate (130 mg, 0.26 mmol) in THF (4 mL) and
deionized H2O (1 mL) was stirred at rt for 16 h, then the organic solvent was evaporated under
reduced pressure. The resultant aqueous solution was acidified to pH 4.5 with 1N HCl (2 mL) at
0 oC and extracted with EtOAc (3 × 10 mL). The organic extracts were washed with brine (10
mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by
SiO2 column chromatography eluting with a gradient of 0-5% IPA/hexanes to afford 20-azido19(S)-(tert-butyldimethylsilyloxy)eicosa-5(Z),14(Z)-dienoic acid (114 mg, 90%) as a colorless oil.
TLC: 60% EtOAc/hexane, Rf ≈ 0.5.
1
H NMR (CDCl3, 400 MHz) δ 5.47–5.25 (m, 4H), 3.79–3.10 (m, 1H), 3.24 (dd, J = 12.4, 4.1 Hz,
1H), 3.13 (dd, J = 12.5, 5.9 Hz, 1H), 2.35 (t, J = 7.5 Hz, 2H), 2.14–1.93 (m, 8H), 1.70 (p, J = 7.5
Hz, 2H), 1.59–1.44 (m, 2H), 1.44–1.23 (m, 12H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H); 13C
NMR (CDCl3, 101 MHz) δ 180.18, 131.30, 130.52, 129.06, 128.12, 71.71, 56.62, 34.65, 33.43,
29.71, 29.67, 29.43, 29.27, 29.26, 27.26, 27.22, 27.16, 26.42, 25.79, 25.22, 24.58, 18.01, -4.63,
-4.66.
O
OH
O O
S
H2N
N3
OTBDMS
O O O
S
N
H
N3
OTBDMS
O
N
H
EDCI HCl
O
O
N
H
O
A mixture of 3-(4-benzoylphenyl)-N-(4-sulfamoylphenyl)propanamide (26 mg, 0.06 mmol),
EDCI·HCl (7 mg, 0.045 mmol), and DMAP (6 mg, 0.045 mmol) was subjected to high vacuum
for 30 min at rt. The mixture was then dissolved in dry DMF (1 mL) and a solution of 20-azido19(S)-(tert-butyldimethylsilyloxy)eicosa-5(Z),14(Z)-dienoic acid (20 mg, 0.04 mmol) in
anhydrous DMF (1 mL) was added. After 12 h, the mixture was charged with water (10 mL) and
4
extracted with EtOAc (3 × 30 mL). The organic layer was washed with water (5 × 2 mL), brine
(10 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was
purified by SiO2 column chromatography eluting with a gradient of 50-70% EtOAc/hexanes to
afford
20-azido-N-((4-(3-(4-benzoylphenyl)propanamido)phenyl)sulfonyl)-19(S)-(tertbutyldimethylsilyloxy)eicosa-5(Z),14(Z)-dienamide (25 mg, 70%) as a waxy solid. TLC: 60%
EtOAc/hexanes, Rf ≈ 0.45.
1
H NMR (500 MHz, CDCl3) δ 8.03–7.99 (m, 2H), 7.96 (s, 1H), 7.82–7.74 (m, 4H), 7.71–7.64 (m,
2H), 7.64–7.57 (m, 1H), 7.52–7.47 (m, 2H), 7.44 (s, 1H), 7.39–7.33 (m, 2H), 5.44–5.28 (m, 3H),
5.23–5.21 (m, 1H), 3.79–3.67 (m, 1H), 3.26 (dd, J = 12.4, 4.1 Hz, 1H), 3.20–3.10 (m, 3H), 2.78
(t, J = 7.5 Hz, 2H), 2.23 (t, J = 7.5 Hz, 2H), 2.05–1.95 (m, 8H), 1.65–1.63 (m, 2H), 1.55–1.20 (m,
2H), 1.36–1.25 (m, 12H), 0.91 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H).
O O O
S
N
H
N3
OTBDMS
O
N
H
TBAF
O O O
S
N
H
N3
OH
O
O
N
H
O
n-Tetrabutylammonium fluoride (TBAF) (27 µL of a 1 M soln in THF, 0.027 mmol) was added to
a stirring 0 °C solution of 20-azido-N-((4-(3-(4-benzoylphenyl)propanamido)phenyl)sulfonyl)19(S)-(tert-butyldimethylsilyloxy)-eicosa-5(Z),14(Z)-dienamide (20 mg, 0.022 mmol) in dry THF
(1.0 mL). After stirring at rt for 24 h, the reaction mixture was quenched with sat. aq. NH4Cl (5
mL) and extracted with EtOAc (3 × 6 mL). The organic extracts were washed with water (1.0
mL), brine (1.0 mL) and dried over Na2SO4, concentrated under reduced pressure and purified
by
PTLC
eluting
with
5
%
MeOH/CH2Cl2
to
give
20-azido-N-((4-(3-(4benzoylphenyl)propanamido)phenyl)sulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide
(15
mg, 86%) as a white waxy solid. TLC: 5% MeOH/CH2Cl2, Rf ≈ 0.45.
1
H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.75 (dd, J = 8.3, 1.4 Hz,
2H), 7.70 (d, J = 8.2 Hz, 2H), 7.63–7.53 (m, 3H), 7.49–7.42 (m, 2H), 7.31 (d, J = 8.2 Hz, 2H),
5.46–5.05 (m, 4H), 3.78–3.68 (m, 1H), 3.35 (dd, J = 12.4, 3.4 Hz, 1H), 3.23 (dd, J = 12.5, 7.3
Hz, 1H), 3.11 (t, J = 7.7 Hz, 2H), 2.77 (t, J = 7.7 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 2.06–1.86 (m,
8H), 1.62–1.54 (m, 2H), 1.48 –1.41 (m, 2H), 1.31–1.18 (m, 12H).
13
C NMR (101 MHz, CDCl3) δ 197.10, 171.19, 170.92, 145.73, 143.12, 137.38, 135.73, 132.86,
132.67, 131.58, 130.71, 130.56, 130.07, 129.54, 128.91, 128.40, 128.36, 127.76, 119.13, 70.83,
57.03, 38.63, 35.64, 33.81, 31.06, 29.58, 29.54, 29.27, 29.15, 29.13, 27.15, 26.91, 26.17, 25.52,
24.19.
a
The preparation of this compound will be reported elsewhere.
Cell Culture. Human microvascular or umbilical vein endothelial cells (Lonza Inc., Allendale NJ)
were used in this study. Both responded equally to 20-HETE with respect to GPR75 activation
and are referred as EC throughout the manuscript. Cells were grown in Medium 131 containing
growth supplement (Invitrogen, Waltham, MA). Passages 2-3 were used for all experiments. For
the aortic vascular smooth muscle cell experiments, A7r5 cells were used (ATCC, Manassas,
5
VA). All cells were maintained at 37oC in a humidified incubator with an atmosphere of 5%
CO2:95% O2. For most experiments, cells were grown in 6-well-plates to 80-90% confluence
and placed in serum-free (Hank’s Buffered Salt Solution, HBSS) media for 12 h prior to addition
of 20-hydroxy-5,8,10,14-eicosatetraenoic acid (20-HETE, 10 nmol/L) with and without the 20HETE antagonist N-(20-hydroxyeicosa-6(Z), 15(Z)-dienoyl) glycine (20-HEDGE). 3
Click-Chemistry. Cell membranes were prepared using the Mem-PER Plus Membrane Protein
Extraction Kit (Thermo Fisher Scientific, Grand Island, NY). Cell extracts (20 µg, 10µl) were
incubated with 10 nmol/L (5 µl) of the photoactive cross linker 20-azido-N-((4-(3-(4benzoylphenyl)propanamido)phenyl)sulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide
(20APheDa) in 6 x 50 mm borosilicate glass culture tubes. Samples were exposed to 365 nm long
wave UV light for 15 min in a Stratalinker 2400 (Stratagene, La Jolla, CA). After
photocrosslinking, 5 µl of 50 µmol/L of dibenzocyclooctyne (DBCO) 800CW Infrared Dye (LICOR, Lincoln, NE) was added to each sample and incubated for 1 h. The DBCO IRDye
preferentially reacts with the azide functional group forming a covalent bond. Loading dye (x4; 5
µl) containing glycerol (40%), Tris/HCl pH 6.8 (240 mM), SDS (8%), bromophenol blue (0.04%)
and beta-mercaptoethanol (5%) was added to each sample (final volume, 25 µl) and samples
were run on a 4-20% Mini-PROTEAN TGX precast gel (Bio-Rad, Hercules, CA). Each sample
contained 5 µl loading dye, 5 Samples did not undergo boiling or changes in temperature in
order to prevent changes in 20-APheDa binding signal. Excess heat has been characterized as
potentially disruptive in several click-compound assays. 4, 5 In-gel sample-complex IRDye signal
was detected using the LI-COR Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and
Odyssey Application Software Version 3.0.21. Upon visualization, gels were marked and
respective bands were extracted, placed in tubes containing 100 μl de-ionized water and sent
for protein sequencing to Applied Biomics (Hayward, CA). Protein identification of the dominant
band location was based on peptide fingerprint mass mapping (using MS data) and peptide
fragmentation mapping (using MS/MS data). The MASCOT search engine was used to identify
proteins from primary sequence databases. Protein partner analysis of the top protein hits were
conducted using STRING and NCBI protein partner databases, For competition experiments, 10
μg of HMVEC cell membranes were incubated with 0.1 nmol/L of 20-APheDa in the presence
and absence of 100 μmol/L 20-HETE or 100 μmol/L 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic
acid (12(S)-HETE) for 15 min prior to UV photocrosslinking and the addition of DBC IRDye. Ingel visualization was assessed using the LI-COR Odyssey Infrared Imaging System (LI-COR,
Lincoln, NE) and Odyssey Application Software Version 3.0.21.
Synthesis of [5,6-;8,9-;11,12-;14,15-3H]20-hydroxyeicosatetraenoic acid [3H8] 20-HETE.
Radiolabeled [3H8] 20-HETE was synthesized as follows. A mixture of 50 μl
[5,6,8,9,11,12,14,15-3H] arachidonic acid (AA) ([50 μCi labeled [3H8] AA at ~50% efficiency = 50
x 106 cpm) (Moravek Biochemicals, Brea, CA) with 7.5 nmols of unlabeled AA (Cayman
Chemicals, Ann Arbor, MI) for a final specific activity of 6 x 103 cpm/pmol was prepared. The AA
mixture was dried under N2 in the incubation vial. The enzyme mixture including reconstituted
20-HETE synthase composed of purified recombinant CYP4A11 0.25 μmol/L, CYP450 OxidoReductase (P450-OR) 2.5 μmol/L and Cytochrome b5 (b5) 2.5 μmol/L (mixed in a 1:10:1 molar
ratio) in the presence of 50 ug/ml dilauroyl-phosphatidylcholine (DLPC) (Sigma, St Louis, MO)
was mixed and incubated for 10 minutes. After the incubation, 170 μl of buffered NADPH
generating mix containing: 50 mmol/L Tris-Cl pH 7.4 containing 10 mmol/L MgCl2 (Sigma, St
Louis, MO), 2 mg/ml Sodium Isocitrate (Sigma, St Louis, MO), 0.2 IU/ml Isocitrate
dehydrogenase (Sigma, St Louis, MO), and 1 mmol/L 1,4 dithio-D-threitol (DTT) (Sigma, St
Louis, MO) was added to the dried AA mixture followed by the addition of the pre-incubated
enzyme mixture. The mixture was kept for 5 minutes at 37o C with gentle mixing. The reaction
was started by adding 10 μl of 20 mmol/L NADPH (EMD Millipore, Billerica, MA) and stopped
6
after 10 min at 370C by the addition of 200 μl of CH3CN 0.5% CH3COH (AcOH) (Sigma, St
Louis, MO). The mixture was vortexed and spun at full speed in a mini-centrifuge. The
supernatant was collected and dried under N2. A pasteur pipette SiO2 column (~2.5 x 0.5 cm)
was made and equilibrated with Hexane/0.5% AcOH. The dried sample was dissolved in 2 ml of
Hexane/0.5% AcOH (Sigma, St Louis, MO) and loaded onto the column. The column was
washed with 10 ml Hexane/0.5% AcOH. The sample was eluted with 5-10 ml of 50% Ethyl
Ether/50% Hexane containing 0.5% AcOH (Sigma, St Louis, MO). The eluted sample was then
dried under N2 and saved in -80oC for HPLC purification. The total CPM in the purified [3H8] 20HETE sample was assessed using a Hidex 300 SL liquid scintillation counter (Hidex, Turku,
Finland).
[3H8] 20-HETE Binding Assays. Cells were harvested by trypsinization followed by
centrifugation and the cell pellet was resuspended in Hanks' balanced salt solution containing
protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). These
experiments followed a similar protocol by Chen et al. 6 Briefly, cells were then sonicated (20-s
bursts) on ice five times using a Q500 Sonicator (QSonica Newton, CT). The homogenate was
centrifuged at 1000g for 10 min at 4°C to remove unbroken cells. The supernatant was
centrifuged at 150,000g for 45 min at 4°C. The pellet represents the membrane fraction and was
resuspended in HBSS containing 50 mmol/L HEPES, pH 7.4. For dissociation studies, 50 μg of
HMVEC membrane fraction was incubated with [3H8] 20-HETE (8000 CPM equivalent to 6.67
nmol/L) in the presence and absence of increasing concentrations of unlabeled 20-HETE or
12(S)-HETE (0.01-100 nmol/L) for 15 min. Incubations were terminated by filtration through
GF/B glass filter paper (Whatman, Clifton, NJ), followed by five washes with 5 ml of ice-cold
binding buffer consisting of 20 mmol/L HEPES, 10 mmol/L CaCl2, and 10 mmol/L MgCl2, at pH
7.4. Radioactivity ([3H8] 20-HETE bound to membrane proteins attached to filter paper) was
assessed using a Hidex 300 SL liquid scintillation counter (Hidex, Turku, Finland).
Transfection with siRNA. Cells were grown on 6-well plates to 70% confluence. Cells were
washed with siRNA transfection medium (Santa Cruz, Dallas, TX) and incubated with a mixture
of GPR75 (sc-94341), GIT1 (sc-35477) or Control (sc-37007) siRNAs (Santa Cruz, Dallas, TX)
and siRNA transfection reagent (Santa Cruz, Dallas, TX) for 6 h following manufacturer’s
instructions. The transfection solutions were replaced with fresh growth medium for an
additional 12 h until 80-90% confluence. Cells were then placed in HBSS for an additional 12 h
and treated with 20-HETE (10 nmol/L) for 5 min for immunoprecipitation studies or 2h for ACE
mRNA expression analysis by real-time PCR. In addition, membranes from control- and
GPR75-specific siRNA-transfected cells were prepared for binding assay as described above.
IP-One assay. IP-1, a downstream metabolite of IP3, accumulates in cells following Gq receptor
activation and has been used as a functional assay for Gq-GPCR. The assay used the IP-One
HTRF assay kit (Cisbio, Beford, MA) and was performed according to the manufacturer’s
instructions. Briefly, cells were seeded into 96-well tissue culture treated white microplates and
grown in regular growth media at 37°C in a 5% CO2:95% O2 atmosphere for 24 h before the
assay. Media was then removed, 20-HETE prepared in stimulation buffer containing LiCl (50
mmol/L) to prevent IP-1 degradation was added and cells were incubated for 5 min. The IP1-d2
conjugate in lysis buffer was added to wells followed by addition of anti-IP1-Cryptate antibody.
Plates were incubated for 1 hour at room temperature and then read on Synergy HT plate
reader (BioTek, Winooski, VT) using excitation at 320 nm and emissions at both 620nm and
665nm. The ratio of 665nm/620nm emissions was calculated. A standard curve of the ratio
665/620 versus log 1P-1 concentration was plotted using a non-linear least squares fit
(sigmoidal dose response variable slope, 4PL) and unknowns were read from standard curve as
nM concentration of IP1.
7
Immunoprecipitation. Cells were cultured on 6-well plates to 80-90% confluence and starved
in serum-free media for 12 h. Cells were treated with 20-HETE (10 nmol/L) or its vehicle (PBS)
for 5 min. In some experiment, cells were incubated with CCL5 (1 nmol/L) for 5 min. Cells were
lysed with 1X RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma, St Louis, MO) containing
protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). Protein
concentrations were determined using the Bradford protein assay (Eppendorf BioPhotometer).
Immunoprecipitation was conducted using the Dynabeads Protein G Immunoprecipitation Kit
(Life Technologies, Grand Island, NY). Dynabeads were incubated with primary antibodies
against human EGFR antibody (AHR5062, Invitrogen, Camarillo, CA, 175 kDa), GPR75 (sc164538, Santa Cruz, Biotechnology, Dallas, TX, 55 kDa), and GIT1 (sc-9657, Santa Cruz,
Biotechnology, Dallas, TX, 95 kDa), for one hour prior to washing and incubation with 5 µg of
HMVEC cell lysate overnight. Samples were then washed and eluted per manufacturer’s
protocol and loaded onto a 4-20% Mini-PROTEAN TGX precast gel (Bio-Rad, Hercules, CA)
and transferred using the Trans-Blot® Turbo™ transfer system to a PVDF membrane.
Immunoblotting for respective associated proteins was conducted using phosphorylated tyrosine
antibody (SC-7020, Santa Cruz, Biotechnology, Dallas, TX), EGFR antibody (AHR5062,
Invitrogen, Camarillo, CA, 175 kDa), GPR75 (sc-164538, Santa Cruz, Biotechnology, Dallas,
TX, 55 kDa), GIT1 (sc-9657, Santa Cruz, Biotechnology, Dallas, TX, 95 kDa), HIC-5 (sc-17616,
Santa Cruz, Biotechnology, Dallas, TX, 48 kDa), G alpha q/11 (sc-392, Santa Cruz,
Biotechnology, Dallas, TX, 40-41 kDa), c-SRC (sc-19, Santa Cruz, Biotechnology, Dallas, TX,
60 kDa). For vascular smooth muscle cell experiments, the PKCα (C-20) sc-208 and MaxiKβ
(FL-191) sc-33608 (Santa Cruz, Biotechnology, Dallas, TX, 80 and 26-37 kDa, respectively)
were used. Membrane fluorescence-based immunodetection was conducted using the
appropriate LI-COR secondary IRDye antibody and LI-COR Odyssey Infrared Imaging System
(LI-COR, Lincoln, NE). Respective band density was quantified using the Odyssey Application
Software Version 3.0.21. The GPR75 antibody was a polyclonal antibody against the c-terminal;
it recognized a single 54-55 KDa protein band as shown in supplementary Fig. 2.
Western Blot Analysis. Samples were lysed with 1X RIPA (Radio-Immunoprecipitation Assay)
buffer (Sigma, St. Louis, MO) containing protease and phosphatase inhibitor cocktails (Roche
Diagnostics, Indianapolis, IN). Protein concentrations were determined using the Bradford
protein assay (Eppendorf BioPhotometer). Samples (20 μg) were run on a 4-20% MiniPROTEAN TGX precast gel (Bio-Rad, Hercules, CA) and transferred using the Trans-Blot®
Turbo™ transfer system to a PVDF membrane. Primary antibodies included: ACE (sc-12184,
Santa Cruz, Biotechnology, Dallas, TX, 195 kDa) and β-Actin monoclonal IgG (Sigma, St Louis,
MO, 42 kDa). Membrane fluorescence-based immunodetection was conducted using the
appropriate LI-COR secondary IRDye antibody and LI-COR Odyssey Infrared Imaging System
(LI-COR, Lincoln, NE). Respective band density was quantified using the Odyssey Application
Software Version 3.0.21.
SYBR Green qPCR and Analysis. cDNAs synthesized from a panel of various C57Bl/6 mouse
tissues previously reported7 were used for the tissue distribution assessment of mouse Gpr75. A
specific primer set of oligonucleotides was designed for the single coding exon of Gpr75. The
forward primer (5’-AAGCAGCCTAATTGTACAGCCT-3’) and the reverse primer (5’TCCCTTCCCCATCCATAAGACT-3’) were each diluted to a concentration of 10 μM, and 0.5 μl
was used for each 10 μl qPCR reaction along with 5 μl of Power SYBR Green PCR Master Mix
(Life Technologies (Carlsbad, CA). Samples were run in triplicate on an Applied Biosystems
ViiA7 Real-Time PCR System (Life Technologies) with PCR conditions used as follows: 50oC for
2 minutes; 95oC for 10 minutes, followed by 40 cycles of 95oC for 15 seconds, then 60oC for 1
minute; a dissociation stage of 95oC for 15 seconds, 60oC for 15 seconds, and 95oC for 15
8
seconds. Data was analyzed using the 2-ΔΔCT method 8 with the mouse tissue expression values
normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Outliers as determined by
the Grubbs Outlier test 9 were removed. The GraphPad Prism 6.0C program (GraphPad
Software, San Diego, CA) was used to generate the tissue expression graph. For determination
of ACE expression in EC, cells were cultured on 6-well plates to 80-90% confluence and starved
in serum-free HBSS media for 24 h and treated with 20-HETE (10 nmol/L) for 2 h in HBSS.
Cells were washed with 1X PBS followed by lysis buffer (Denville Scientific, Metuchen, NJ).
Total RNA was isolated using the SpinSmart RNA Purification kit (Denville Scientific, Metuchen,
NJ) and quantified using the Synergy HT Take3 Microplate Reader (BioTek,Winooski, VT). RT
reaction of total RNA (500 ng) was performed using the qScript cDNA Synthesis Kit (Quanta
Biosciences, Gaithersburg, MD). Sequences for PCR primers used are as follows: angiotensin
converting enzyme (ACE) sense: 5’- TCC CAT CCT TTC TCC CAT TTC -3’; ACE antisense: 5’GCT CTC CCA ACA CCA CAT TA -3’; 18S rRNA sense: 5’- GAT GGG CGG CGG AAA ATA G
-3’; 18S rRNA antisense: 5’- GCG TGG ATT CTG CAT AAT GGT - 3’. Quantitative Real-Time
PCR was performed using the PerfeCTa SYBR Green FastMix Low ROX Kit (Quanta
Biosciences, Gaithersburg, MD) and the Mx3000p Real-Time PCR System (Stratagene, Santa
Clara, CA) as previously described.10
TaqMan qPCR. The Mm00558537_s1 Gpr75 TaqMan primer/probe and the Mm99999915_g1
Gapdh primer/probe were purchased from Life Technologies. For all qPCR reactions 0.5 μl of
the 20X primer/probes and 5 μl of 2X Maxima Probe/ROX qPCR Master Mix (Thermo Scientific,
Pittsburgh, PA) were used in a 10 μl final reaction volume. 1 μl of 1 ηg/μl diluted tissue cDNAs
were used for each reaction. The PCR conditions were as follows: 50oC for 2 minutes; 95oC for
10 minutes, followed by 40 repeat cycles of 95oC for 15 seconds, and 60oC for 1 minute.
Samples were run in triplicate.
Animal Studies. All experimental protocols were approved by the Institutional Animal Care and
Use Committee (IACUC) and by the Institutional Biosafety Committee. The generation and
phenotypic characterization of the Cyp4a12 transgenic mice in which the expression of the
Cyp4a12-20-HETE synthase is under the control of an androgen-independent, tetracycline
(doxycycline, DOX)-sensitive promoter have been previously described 11. Male Cyp4a12tg
mice (8-14-week-old) were used in all experiments. DOX (1 mg/ml drinking water) was
administered to promote the activation of the sole murine 20-HETE synthase, Cyp4a12. Blood
pressure was monitored by radiotelemetry and tail cuff before and after administration of DOX
for 14-35 days as described below. At the end of all experiments, mice were anesthetized with
ketamine (70 mg/kg) and xylazine (70 mg/kg) and laporotomy was performed. Renal
preglomerular arteries (the whole vascular tree) or segments of the interlobar arteries (~80-100
µm) were microdissected and collected for western blot analysis and functional studies.
GPR75 SMARTvector Lentiviral shRNA Studies. All lentiviral constructs were made by
Dharmacon (GE). The SMARTvector Lentiviral shRNA used included non-targeting hCMVTurboGFP shRNA (VSC7420) and lentiviral hCMV-TurboGFP shRNA particles targeted at
GPR75 (V3SM7603 Seq: GGG TTG GCT TCA TGG TTT C). Cyp4a12tg mice were
anesthetized under isoflurane and administered lentiviral constructs via a single retro-orbital
injection to maximize systemic distribution.12 Each mouse was injected with 100 µl of either
4.33x109 TU/mL of non-targeting or 7.29x109 TU/mL of GPR75-targeted shRNA particles. The
administration of DOX (1 mg/ml) was initiated 48 h after the lentiviral injection. For western
blotting, renal preglomerular microvessels were collected and lysed with 1X RIPA buffer (Sigma,
St Louis, MO) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics,
Indianapolis, IN). Protein concentrations were determined using the Bradford protein assay
(Eppendorf BioPhotometer). Protein samples (20 µg) were loaded onto a 4-20% Mini-
9
PROTEAN TGX precast gel (Bio-Rad, Hercules, CA) with respective loaded EZ-Run Prestained
Rec Protein Ladder (Fisher BioReagents, Waltham, MA) markers. SDS-polyacrylamide gels
were transferred to Tran-Blot Turbo Mini PVDF membranes (Bio-Rad, Hercules, CA) followed
by blocking buffer (Li-Cor, Lincoln, NE) and subsequent incubation with primary and secondary
antibodies.
Immunofluorescence Imaging. Liver tissue from mice receiving non-targeting lentiviral hCMVTurboGFP shRNA particles was harvested to assess GFP transduction. Kidney tissue from
C57BL/6 was harvested to examine vascular endothelial GPR75 expression. Tissue samples
were fixed in 4% paraformaldehyde at 4°C. Tissues were embedded in cassettes using TissueTek Optimum Cutting Temperature compound. Tissue sections were cut at 3-4 µm thickness.
Goat anti GPR75 (sc-164538) and rabbit anti-CD31 antibodies were used as primary antibodies
and were from Santa Cruz, Biotechnology, Dallas, TX. Alexa Fluor® 555-conjugated donkey
anti-goat and Alexa Fluor® 488-conjugated goat anti-rabbit IgG were used as secondary
antibodies and were from Abcam (Cambridge, MA). Immunofluorescence of GFP, GPR75 and
CD31 was visualized using a Zeiss Axio Imager M1 fluorescent microscope. Images were
captured using AxioVision multichannel image processing software.
Blood Pressure Measurements. Systolic blood pressure measurements in Cyp4a12tg mice
were taken using the CODA tail-cuff system (Kent Scientific, Torrington, CT), which utilizes
volume pressure recording sensor technology. Mice were acclimated to the machine for one
week prior to day 0 and blood pressure was monitored throughout the length of the experiment.
Values within ± 10% of their mean blood pressure measurements were obtained. In one set of
experiments (n=1/group), blood pressure measurements were conducted in the conscious state
by radio-telemetry without distress to the animal after post-surgical recovery. The radiotelemetric system from Data Science International (DSI, St Paul, MN) was used for this
procedure. Systolic blood pressure was continuously acquired by implantation of telemetric
probe (model TA11PA-C10) into the aorta via the left carotid artery. After one week of recovery
from the surgical procedure, blood pressure readings were recorded every ten minutes using
Dataquest ART software purchased from Data Science International (DSI). Baseline blood
pressure was collected for four days prior to and during the administration of DOX (online
Figure V).
Measurements of Vascular Function. Microdissected renal interlobar arteries were mounted
on wires in the chambers of a multivessel myograph (JP Trading, Aarhus, Denmark) filled with
Krebs buffer (37°C) gassed with 95% O2 / 5% CO2. For 20-APheDa studies Sprague-Dawley
(SD) rats were used. After mounting and 30-60 min of equilibration, the vessels were set to an
internal circumference equivalent to 90% of that which they would have in vitro when placed
under a transmural pressure of 100 mmHg. Isometric tension was monitored continuously
before and after the experimental interventions. A cumulative concentration-response curve to
phenylephrine (1x10-9-1x10-4 mol/L) was constructed. Additional experiments were conducted to
determine concentration-response curves for acetylcholine-induced relaxation after maximal
contraction. A cumulative relaxation-response curve to acetylcholine (1x10-8-1x10-4 mol/L) was
constructed and the maximal relaxation was recorded. Wire myograph was used to measure
constrictor responses to phenylephrine (10-9 to 5x10-5 mol/L) and relaxation to acetylcholine (109
to 5x10-5 mol/L) of renal interlobar arteries (~100 µm diameter).
Measurements of Media Thickness, Media to Lumen Ratio and Medial Cross-Sectional
Area. Assessment of remodeling was performed by pressure myography according Schiffrin &
Hayoz 13 and as previously described.14 Briefly, segments of renal interlobar arteries were
dissected and mounted on a pressurized myograph and equilibrated for 1 h in oxygenated Ca2+-
10
free Krebs buffer at 37°C. The operator was blinded to treatments except for the blood pressure
range of the animal. Lumen diameters from normotensive animals were determined at 100
mmHg and hypertensive animals at 140 mmHg. Measurements of outer diameter (OD) and
inner diameter (ID) under passive conditions were used to calculate media thickness [(OD −
ID)/2], media to lumen ratio (OD − ID)/ID], and medial cross-sectional area [CSA = (π/4) ×
(OD2 − ID2)].
Statistical Analysis Data are expressed as mean ± SEM. Significance of difference in mean
values was determined using t test and 1-way ANOVA, followed by the Newman-Keul post hoc
test. P<0.05 was considered to be significant.
REFERENCES
1. Broemer M, Krappmann D and Scheidereit C. Requirement of Hsp90 activity for IkappaB
kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kappaB activation.
Oncogene. 2004;23:5378-86.
2. Cisneros JA, Bjorklund E, Gonzalez-Gil I, Hu Y, Canales A, Medrano FJ, Romero A, OrtegaGutierrez S, Fowler CJ and Lopez-Rodriguez ML. Structure-activity relationship of a new
series of reversible dual monoacylglycerol lipase/fatty acid amide hydrolase inhibitors. J Med
Chem. 2012;55:824-36.
3. Wu CC, Mei S, Cheng J, Ding Y, Weidenhammer A, Garcia V, Zhang F, Gotlinger K,
Manthati VL, Falck JR, Capdevila JH and Schwartzman ML. Androgen-sensitive
hypertension associates with upregulated vascular CYP4A12-20-HETE synthase. J Am Soc
Nephrol. 2013;24:1288-96.
4. Martin BR. Nonradioactive analysis of dynamic protein palmitoylation. Current protocols in
protein science. 2013;73:Unit 14.15.
5. Mackinnon AL and Taunton J. Target Identification by Diazirine Photo-Cross-linking and
Click Chemistry. Current protocols in chemical biology. 2009;1:55-73.
6. Chen Y, Falck JR, Manthati VL, Jat JL and Campbell WB. 20-Iodo-14,15-epoxyeicosa-8(Z)enoyl-3-azidophenylsulfonamide: photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid
receptor. Biochemistry. 2011;50:3840-8.
7. Graves JP, Gruzdev A, Bradbury JA, DeGraff LM, Li H, House JS, Hoopes SL, Edin ML and
Zeldin DC. Quantitative Polymerase Chain Reaction Analysis of the Mouse Cyp2j Subfamily:
Tissue Distribution and Regulation. Drug metabolism and disposition: the biological fate of
chemicals. 2015;43:1169-80.
8. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif).
2001;25:402-8.
9. Grubbs EF. Sample Criteria for Testing Outlying Observations. Ann Math Statist.
1950;21:27-58.
10. Garcia V, Shkolnik B, Milhau L, Falck JR and Schwartzman ML. 20-HETE Activates the
Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-kappaB Translocation
and Promoter Binding. J Pharmacol Exp Ther. 2016;356:525-33.
11. Wu CC, Mei S, Cheng J, Ding Y, Weidenhammer A, Garcia V, Zhang F, Gotlinger K,
Manthati VL, Falck JR, Capdevila JH and Schwartzman ML. Androgen-Sensitive
Hypertension Associates with Upregulated Vascular CYP4A12-20-HETE Synthase. J Am
Soc Nephrol. 2013.
12. van den Brand BT, Vermeij EA, Waterborg CE, Arntz OJ, Kracht M, Bennink MB, van den
Berg WB and van de Loo FA. Intravenous delivery of HIV-based lentiviral vectors
11
preferentially transduces F4/80+ and Ly-6C+ cells in spleen, important target cells in
autoimmune arthritis. PloS one. 2013;8:e55356.
13. Schiffrin EL and Hayoz D. How to assess vascular remodelling in small and medium-sized
muscular arteries in humans. Journal of hypertension. 1997;15:571-84.
14. Garcia V, Joseph G, Shkolnik B, Ding Y, Zhang FF, Gotlinger K, Falck JR, Dakarapu R,
Capdevila JH, Bernstein KE and Schwartzman ML. Angiotensin II receptor blockade or
deletion of vascular endothelial ACE does not prevent vascular dysfunction and remodeling
in 20-HETE-dependent hypertension. Am J Physiol Regul Integr Comp Physiol.
2015;309:R71-8.
12
FIGURE LEGENDS
Figure I: Schematic depicting the click-chemistry, retrieved proteomics data and target
identification. The protein peptide summary represents the most frequent hits from proteomics
analyses of six separate in-gel 20-APheDa-EC complex. The line between the marker lane (M)
and the ECm lane reflects the fact that although the lanes are part of the same gel they are on a
separate infrared channel.
Figure II: Tissue distribution of Gpr75 expression in C57Bl/6 mouse tissues. cDNAs
synthesized from a panel of various C57Bl/6 mouse tissues and Gpr75 mRNA levels were
quantified by SYBR Green qPCR (A) and by TaqMan (B) using specific Gpr75 primers. Results
are mean±SD, n=6 for all tissues except ovary, testis and uterus, n=3. Samples were run in
triplicate.
Figure III: A representative immunoblot showing a single GPR75 immunoreactive band of ~55
kDa in EC lysate using the anti-GPR75 polyclonal antibody.
Figure IV: Effect of CCL5 on a) GPR75-Gαq/11 association, b) EGFR phosphorylation and c)
ACE mRNA in EC. EC were incubated with CCL5 (1 nmol/L) for 5 min and cells were harvested
and processed for immunoprecipitation of either GPR75 or EGFR followed by immunoblotting
for (A) Gαq/11 and (B) phospho-tyrosine. For ACE mRNA (C), cells were incubated with CCL5 (1
nmol/L) for 2 h and processed for real-time PCR analysis of ACE mRNA. Results are
mean±SEM, n=3.
Figure V: Representative radio-telemetric blood pressure measurements in a Cyp4a12tg
mouse treated with either control or GPR75 shRNA and receiving DOX (n=1/group). A) Mean
Arterial Pressure, B) Systolic Blood Pressure, C) Diastolic Blood Pressure and D) Heart Rate.
Figure VI: GPR75 protein levels in the liver (A) and heart (B) of mice receiving a bolus injection
of SMARTvector Control shRNA or SMARTvector GPR75 shRNA (retro-orbital injection of 100
µl of 4.33x109 TU/mL of non-targeting or 7.29x109 TU/mL of GPR75-targeted shRNA particles,
respectively). Tissues were harvested 35 days after injection and processed for measurements
of GPR75 levels by Western blot (n=3; mean±SEM; *p<0.05)
On line Figure I
Click
Chemistry
kDa M
170
130
95
72
55
43
34
26
Protein Peptide
Summary
Interactome
Tracing
Orphan Receptor
Protein Partner
Analysis
ECm
Ankyrin
HIC-5
Zinc Finger Protein
Plasminogen-like
Protein A
Hyaluronan Synthase
GTP-BindingProtein
Vimentin
Keratin
Actin
Orphan
Receptor
Candidate
HIC-5
GIT1
17
Target Protein Confirmation
siRNA/shRNA
GPR75
On line Figure II
A
B
(SYBR Green)
(TaqMan)
On line Figure III
IP: GPR75
kDa
IB:GPR75
M
EC
170
130
95
72
55
43
34
26
GPR75
On line Figure IV
A
IP: GPR75
Gαq/11
Vehicle
- 55
(Relative to Vehicle)
GPR75
1.5
Gαq/11/GPR75
B
CCL5 - 43
1.0
0.5
0.0
IP: EGFR
P-Tyr
Vehicle
Vehicle
CCL5
CCL5
- 170
P-Tyr/EGFR
(Relative to Vehicle)
EGFR
- 170
1.5
1.0
0.5
0.0
C
Vehicle
CCL5
Vehicle
CCL5
ACE mRNA
(Relative to Vehicle)
2.0
1.5
1.0
0.5
0.0
C
Diastolic Blood Pressure
(mmHg)
Systolic Blood Pressure
(mmHg)
Mean Arterial Pressure
(mmHg)
A
Heart Rate
(BPM)
On line Figure V
shRNA Inj
DOX
B
Days
shRNA Inj
DOX
Days
shRNA Inj
DOX
D
Days
shRNA Inj
DOX
Days
On line Figure VI
B
A
Control
shRNA
- 55
1.2
0.8
*
0.4
Control
shRNA
GPR75
shRNA
- 55
α-tubulin
GPR75 / α-tubulin
(relative to control)
- 43
β-Actin
GPR75 / β-Actin
(relative to control)
GPR75
shRNA
- 55 GPR75
GPR75
0.0
Control
shRNA
GPR75
shRNA
1.2
0.8
0.4
0.0
*
Control
shRNA
GPR75
shRNA