Am J Physiol Cell Physiol 310: C942–C954, 2016.
First published April 13, 2016; doi:10.1152/ajpcell.00275.2015.
Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from
lacrimal acini in a mouse model of Sjögren’s Syndrome
Zhen Meng,1 Maria C. Edman,2 Pang-Yu Hsueh,1 Chiao-Yu Chen,1 Wannita Klinngam,1
Tanya Tolmachova,3 Curtis T. Okamoto,1 and Sarah F. Hamm-Alvarez1,2
1
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los
Angeles, California; 2Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los
Angeles, California; 3Imperial College London, London, United Kingdom
Submitted 18 September 2015; accepted in final form 6 April 2016
Rab; lacrimal gland; exocytosis; lysosomal protease; Sjögren’s
Syndrome; tears
(LG) is an exocrine gland responsible
for the secretion of lacrimal fluid representing the main portion
of the tears. Lacrimal fluid is a highly complex mixture of
water, electrolytes, and proteins, including antibodies, hydrolases, growth factors, cytokines (53), and proteases and their
inhibitors (10). The LG is composed primarily of acinar cells
(LGAC), which constitute ⬃80% of its total mass, with the
remaining 20% comprising ductal cells, myoepithelial cells,
and lymphocytes (12). The principal function of LGAC, secretory epithelial cells with distinct apical and basolateral plasma
THE LACRIMAL GLAND
Address for reprint requests and other correspondence: S. Hamm-Alvarez,
Dept. of Ophthalmology, Univ. of Southern California, 1450 San Pablo St.,
#4900, Los Angeles, CA 90033 (e-mail: [email protected])
C942
membranes, is regulated exocytosis of secretory vesicles (SV)
containing tear proteins.
Dysfunction of the LG is a major factor leading to dry eye,
the most common cause of patient visits to eye care specialists.
Dry eye caused by LG dysfunction is classified as aqueous
deficient, with one of its most severe forms associated with
Sjögren’s Syndrome (SS), a chronic autoimmune disease characterized by lymphocytic infiltration of LG and salivary
glands, leading to severe corneal damage and compromised
oral health. In addition to exocrine gland morbidity, patients
with SS experience weight loss, fatigue, systemic inflammation
of internal organs and, in 5% of patients, B-cell lymphoma
(35). Although SS affects ⬃0.5–1.0% of the general population
(30), its diagnosis is often delayed because of an overlap of its
symptoms with those caused by other autoimmune diseases
and eye diseases, as well as other conditions such as menopause and drug side effects (34). SS diagnosis is further
complicated because of its diverse etiology, driven by environmental, genetic, and hormonal factors (30). Surprisingly, there
are no specific therapies for SS because of the lack of understanding of the mechanisms in the exocrine glands that trigger
tissue-specific inflammation. Current treatments are centered
on alleviation of ocular symptoms by artificial tears combined
with systemic immunomodulatory therapies to control autoimmune responses, rather than targeting the underlying exocrinopathies (14), thus representing a critical barrier to effective
treatment of patients.
To address some of the challenges in diagnosis and treatment of SS, we have studied the early changes in the LG
associated with the onset of autoimmune infiltration (autoimmune dacryoadenitis) characteristic of SS, using the nonobese
diabetic (NOD) mouse model. The NOD mouse is a wellcharacterized spontaneous model of SS, with the classical
clinical manifestations of autoimmune dacryoadenitis detected
in the males from 6 wk of age (23). Our previous work in the
NOD mouse LG revealed that increased expression and activity of cathepsin S (CTSS), a lysosomal cysteine protease,
paralleled the onset of lymphocytic infiltration (28, 51). In
addition to its established role in proteolysis in lysosomes and
remodeling of extracellular matrix, an additional major role of
CTSS is processing of the myosin heavy chain class IImediated antigen presentation in the immune system, including
cleavage of invariant chain and generation of antigenic peptide
(21, 37). Intriguingly, analysis of the LGAC revealed that
much of the increased CTSS protein was, not only present in
immune cells, but also substantially increased in the LGAC,
specifically in apparent mature SV, a change accompanied by
increased CTSS abundance and activity in the tears of the NOD
0363-6143/16 Copyright © 2016 the American Physiological Society
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Meng Z, Edman MC, Hsueh P-Y, Chen C-Y, Klinngam W,
Tolmachova T, Okamoto CT, Hamm-Alvarez SF. Imbalanced
Rab3D versus Rab27 increases cathepsin S secretion from lacrimal
acini in a mouse model of Sjögren’s Syndrome. Am J Physiol Cell
Physiol 310: C942–C954, 2016. First published April 13, 2016;
doi:10.1152/ajpcell.00275.2015.—The mechanism responsible for the
altered spectrum of tear proteins secreted by lacrimal gland acinar
cells (LGAC) in patients with Sjögren’s Syndrome (SS) remains
unknown. We have previously identified increased cathepsin S
(CTSS) activity as a unique characteristic of tears of patients with SS.
Here, we investigated the role of Rab3D, Rab27a, and Rab27b
proteins in the enhanced release of CTSS from LGAC. Similar to
patients with SS and to the male nonobese diabetic (NOD) mouse
model of SS, CTSS activity was elevated in tears of mice lacking
Rab3D. Findings of lower gene expression and altered localization of
Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing
excess CTSS release under physiological conditions. However, CTSS
activity was significantly reduced in tears of mice lacking Rab27
isoforms. The reliance of CTSS secretion on Rab27 activity was
supported by in vitro findings that newly synthesized CTSS was
detected in and secreted from Rab27-enriched secretory vesicles and
that expression of dominant negative Rab27b reduced carbacholstimulated secretion of CTSS in cultured LGAC. High-resolution
3D-structured illumination microscopy revealed microdomains of
Rab3D and Rab27 isoforms on the same secretory vesicles but present
in different proportions on different vesicles, suggesting that changes
in their relative association with secretory vesicles may tailor the
vesicle contents. We propose that a loss of Rab3D from secretory
vesicles, leading to disproportionate Rab27-to-Rab3D activity, may
contribute to the enhanced release of CTSS in tears of patients
with SS.
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
Rab27 isoforms. Contrarily, the release of secretory component
(SC), linked previously to polymeric immunoglobulin receptor
(pIgR) sequestered in Rab3D-enriched SV, into tears was
significantly decreased in 3DKO mice but increased in 27KO.
Our analysis of Rab3D and Rab27 isoform association with SV
in LGAC by confocal fluorescence microscopy (CFM)
showed, as we previously reported (7), significant colocalization of these Rabs on the same SV. However, the enhanced
resolution provided by 3D-structured illumination microscopy
(3D-SIM) revealed that these Rabs were clustered in different
microdomains of the SV and that the relative abundance of
each on any individual SV varied across vesicles. Reduced
release of CTSS activity into tears of mice lacking Rab27
isoforms and into the medium of LGAC expressing dominant
negative (DN) Rab27b suggests that Rab27 isoforms are involved normally in recruitment and/or exocytosis of CTSS.
Conversely, in the 3DKO mouse as well as in the NOD disease
model, as also possibly in SS, a lack of Rab3D recruitment to
SV may result in the predominance of Rab27-mediated events
in exocytosis that recruit and release more of the specific
lysosomal cargo proteins that it recruits, including CTSS.
MATERIALS AND METHODS
Reagents. Carbachol (CCh) was from Sigma-Aldrich (St. Louis,
MO), optimal cutting temperature (O.C.T.) compound from VWR
(Radnor, PA), and Matrigel was from Collaborative Biochemicals
(Bedford, MA). Mouse anti-Rab27a monoclonal and rabbit antiRab27b polyclonal antibodies were from Abcam (Cambridge, United
Kingdom) and Synaptic Systems (Goettingen, Germany), respectively. Rhodamine phalloidin, Alexa Fluor 647 phalloidin,
LysoTracker Red DND-99, secondary antibodies for immunofluorescence (Alexa Fluor 488 goat anti-rabbit; Alexa Fluor 568 goat antirabbit), and the ProLong Antifade Kit were from Invitrogen (Grand
Island, NY). Rabbit polyclonal antibody to recombinant Rab3D was
generated by Antibodies (Davis, CA) as previously reported (13).
Anti-pIgR/SC polyclonal antibody was also generated in rabbits by
Antibodies against the extracellular domain of mouse recombinant
pIgR that is cleaved to form SC and that was expressed in Escherichia
coli, then purified by ammonium sulfate precipitation followed by ion
exchange chromatography. IRDye800-conjugated rabbit IgG F(c)
antibody was from Rockland Immunochemicals (Limerick, PA). The
CTSS Activity Assay Kit was from Biovision (Milpita, CA). The
substrate of ␤-hex, 4-methylumbelliferyl N-acetyl-␤-D-glucosaminide, was from Sigma-Aldrich. The Bio-Rad Protein Assay dye reagent
concentrate was from Bio-Rad Laboratories (Hercules, CA). RNA
extraction kits were obtained from Qiagen (Germantown, MD). The
reverse transcription kit, primers, and master mix for real-time PCR
were purchased from Applied Biosystems (Grand Island, NY). All
other chemicals were reagent grade and obtained from standard
suppliers.
Mice. Several mouse strains were used for these studies. 3DKO
breeding pairs generated as previously described (36) were kindly
provided by Dr. Dietmar Riedel (Max Planck Institute for Biophysical
Chemistry, Göttingen, Germany). The 27KO mice were generated as
previously described (47). Ashen and 27bKO mice were generated by
backcrossing 27KO into the C57BL/6 (C57) background. C57 and
BALB/c mice were obtained from Jackson Laboratories (Sacramento,
CA) or Charles River (Wilmington, MA) or bred in house from
breeding pairs from the same facilities. NOD mice breeding pairs
were from Taconic (Oxnard, CA), and animals used in this study were
from colonies bred in house. Because the major autoimmune
dacryoadenitis is manifested in the male NOD mouse at 12 wk, all
mouse work utilized male mice and their control strains aged 12 wk.
All animal procedures were in accordance with the Guiding Principles
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mouse (28). The mechanisms responsible for increased Ctss
gene expression as well as the recovery of increased CTSS
protein into mature SV were not resolved in this study. However, we recently confirmed that CTSS activity is significantly
enhanced in tears of patients with SS relative to tears of
patients with other dry eye conditions or non-SS autoimmune
diseases (18). Thus tear CTSS represents a potential diagnostic
biomarker for the disease and an early indicator of LG dysfunction. One study in salivary gland has suggested that increased CTSS in salivary gland acinar cells is associated with
conversion of these epithelial cells to function as antigenpresenting cells (41). Therefore, an increased understanding of
the mechanisms of increased CTSS release into tears as well as
other features of its intracellular trafficking is of direct clinical
relevance.
Regulated exocytosis from SV is controlled by multiple
signaling pathways and mediated by numerous molecular effectors. Rab proteins, constituting the largest group in the Ras
superfamily of low-molecular-weight GTPases, are critical
regulators of exocytosis. Rab proteins are synthesized and
present in the cytosol in soluble form and are modified by
addition of geranylgeranyl groups, enabling their association
with membranes. Rabs cycle between active GTP-bound and
inactive GFP-bound states, which are controlled by guanine
nucleotide exchange factors and GTPase activating proteins
(11). The Rab3 and Rab27 subfamilies are closely related and
are both associated with secretory granules or SV in diverse
cell types including those of neural, endocrine, exocrine, and
immune origin (16). Within the Rab3 and Rab27 families,
Rab3D, Rab27a, and Rab27b, have been shown to mediate
regulated exocytosis in exocrine tissues and, specifically, in
LGAC (6, 7, 13, 49). Because of its localization on secretory
granules and its redistribution upon stimulated secretion in a
number of exocrine cells including LGAC (4, 49), Rab3D is
thought to play a key role during regulated exocytosis in
exocrine secretion. Rab27a is expressed and undergoes regulated exocytosis in some exocrine cells, including zymogen
granules in pancreatic cells (38) and SV in parotid acinar cells
(24). Rab27a is also present in LGAC in apparent association
with SV but was not directly implicated in our previous work
in SV formation or maturation (7) and may play a later role in
mature SV trafficking similar to its function in platelets (47).
Rab27b regulates exocytosis of secretory granules such as
amylase-containing secretory granules in parotid gland acinar
cells (24), zymogen granules in pancreatic acinar cells (5), and
mature SV in LGAC (7).
In this study, we found that the increased CTSS activity in
tears of the NOD mouse is not a feature shared by other
lysosomal proteins such as ␤-hexosaminidase (␤-hex) that are
also secreted into tears. We further identified a redistribution of
Rab3D from vesicles located at the subapical region to those
more basolaterally located in NOD mouse LGAC, but no
accompanying change in Rab27b vesicular association, suggesting that altered functioning or recruitment of Rab3D or an
imbalance in Rab3D/Rab27 association with SV characterizes
this disease model. Using Rab3D knockout (3DKO) mice and
mice lacking Rab27a (ashen), Rab27b (27bKO), or both Rab27
isoforms (27KO), we measured the secretion of these same
lysosomal proteins into tears. Intriguingly, CTSS activity was
significantly increased in tears from the 3DKO mouse but
significantly reduced in tears of mice lacking either or both
C943
C944
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
cloned into the pAcGFP1-N1 vector (Clontech, Mountain View, CA)
at the 591-bp (5=end) and the 645-bp (3=end) sites of this vector. The
pAcGFP1-N1 CTSS was digested with restriction endonucleases,
NheI and KpnI, to generate a linearized DNA fragment encoding
CTSS-GFP, which was further subcloned into the pTRE-shuttle2
vector. The pTRE-shuttle2 CTSS-GFP was then digested with restriction endonucleases, I-CeuI and PI-SceI, and ligated into the Adeno-X
system 1 viral DNA provided by the Adeno-X Tet-on expression
system 1 kit (Clontech). The recombinant Ad DNA was linearized by
the restriction endonuclease, PacI, to expose its Ad inverted terminal
repeats before it was transfected into QBI-HEK 293 cells (Qbiogene,
Adenovirus Technology, Carlsbad, CA) for viral assembly and packaging. All procedures used to generate recombinant and replicationdefective Ad were in accordance with the manufacturer’s manual.
Restriction enzymes used were purchased from New England
BioLaboratories (Ipswich, MA). All recombinant vectors used for this
construction were confirmed by DNA sequencing and DNA diagnostic digestion.
The Ad yellow fluorescent protein (YFP)-Rab27b full-length (wildtype, WT), N133I (DN), and Q78L (constitutively active, CA) used in
our previous studies (7) were kind gifts of Dr. Serhan Karvar (University of South Carolina, Columbia, SC) (44). Ad constructs encoding (His)6 epitope-tagged forms of Rab3D full-length (WT), T36N
(DN), and Q81L (CA) were gifts from Dr. John A Williams (University of Michigan, Ann Arbor, MI). Ad CTSS-GFP requires cotransduction with Adeno-X Tet-On and doxycycline induction for protein
expression. The Adeno-X Tet-On regulatory virus encodes a regulatory protein that recognizes the reverse Tet repressor in our constructs
and, therefore, induces the expression of recombinant proteins in
LGAC. The Adeno-X Tet-On regulatory virus was thus also amplified
for use. As for Ad CTSS-GFP, these constructs were amplified in
QBI-HEK 293 cells until the cells showed the characteristic cytopathological effect. Cells were then harvested, and Ad was purified
using cesium chloride gradient ultracentrifugation, while titers were
measured as plaque-forming units by the formation of viral plaques in
sequential dilutions (7).
Preparation of rabbit primary LGAC. Isolation of rabbit LGAC
was as previously described (39). Female New Zealand White rabbits
(1.8 –2.2 kg) were obtained from Irish Farms (Norco, CA) for isolation of LG. Cells isolated from rabbit LG and cultured for 2 days
aggregate into acinus-like structures. These cells display distinct
apical and basolateral domains and maintain a secretory response,
which structurally and functionally mimics the LGAC in vivo (9, 17,
25). These reconstituted rabbit LGAC have been routinely used for all
in vitro work for many years by our laboratory (7, 25, 29) because the
larger size of the rabbit LG relative to the mouse LG (400 mg vs. 10
mg) results in a much greater yield of the acinar cells required for
these studies and enables us to significantly reduce our animal use.
The association of Rab proteins with SV in both systems is comparable (7). For imaging of fixed cells, isolated cells were seeded on
coverslips coated with Matrigel (MatTek, Ashland, MA) in 12-well
plates at 2 ⫻ 106 cells per well. For imaging of live cells, cells were
seeded in 35-mm glass-bottomed Petri dishes coated with Matrigel at
6 ⫻ 106 cells per dish.
For analysis of CTSS-GFP expression and distribution, LGAC
were cotransduced after 48 h of culture. LGAC were coincubated for
2 h at 37°C with Ad CTSS-GFP and the Adeno-X Tet-On at a
multiplicity of infection (MOI) of 4 – 6. Doxycycline was added at a
concentration of 1 ␮g/ml after the removal of virus and replacement
of culture medium. Ad YFP-Rab27b, which does not require doxycycline induction, was similarly transduced alone into LGAC at MOI
of 4 – 6. After a change of fresh medium, LGAC were cultured for
12–20 h for optimal protein expression.
For measuring CTSS and ␤-hex secretion, LGAC were transduced
with Ad YFP-Rab27b full-length, N133I, and Q78L or Ad (His)6
epitope-tagged Rab3D full-length, T36N, and Q81L constructs.
LGAC were incubated with individual Ad constructs for 2 h and
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for the Use of Animals in Research and approved by USC Institutional
Animal Care and Use Committee.
Measurement of enzyme activities in tear and LG lysates. Tear
collection and tear protein activity assays were conducted as described
previously (28). Briefly, the LG was stimulated by adding the agonist
CCh (3 ␮l, 50 ␮M) topically to the gland, and tear fluid was collected
by applying a 2-␮l microcaps pipette (Drummond, Broomall, PA) at
the lateral canthus of the eye, for 5 min. Each LG was stimulated three
times, resulting in a total collection time of 15 min. For preparation of
lysates, LG were collected and homogenized with BeadBlaster 24
Microtube Homogenizer (Benchmark Scientific, Edison, NJ), and the
homogenate was clarified by centrifugation at 10,000 g at 4°C for 5
min. CTSS activity in tear fluid and LG lysates was determined with
the assay kits described herein, according to the manufacturer’s
instructions, and the enzymatic reaction was incubated at 37°C for 2
h. The quantity of the resulting fluorescent products was measured in
a microplate spectrofluorometer (SpectraMax Gemini Plate Reader;
Molecular Devices, Sunnyvale, CA) with 400/505-nm excitation/
emission filters. ␤-hex activity assay procedures were conducted as
described previously (1, 9), and reactions were incubated at room
temperature for 2 h before being read with 365/460-nm excitation/
emission filters (GENios Plus Fluorescence Absorbance Reader;
Tecan, Mannedorf, Germany). Activities of CTSS and ␤-hex were
measured as relative fluorescence units per microgram protein. Total
tear protein concentration was measured using the Bio-Rad protein
assay and expressed as microgram proteins per microliter of tear fluid.
SC secretion was measured by Western blotting with appropriate
primary and secondary antibodies. Equivalent amounts of tear protein
were loaded for each sample. The membrane was scanned, and signal
was quantified by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Immunofluorescence labeling of cells and LG tissue and imaging
by CFM or 3D-SIM. LG from 11–13-wk-old male mice were retrieved
and fixed in 4% paraformaldehyde for 2–3 h and thereafter immersed
in 30% sucrose at 4°C overnight. Fixed LG were embedded in O.C.T.
compound and flash frozen in liquid nitrogen. The blocks were
cryosectioned at 5-␮m thickness and mounted on glass slides. The
sections were permeabilized with 0.1% Triton X-100 for 10 min and
then 1% SDS for 5 min. Adenovirus (Ad)-transduced LGAC were
fixed and permeabilized with ⫺20°C acetone for 20 min. After being
blocked with 1% BSA for 1 h, tissue sections and cells were incubated
with primary and secondary antibodies. After each incubation, samples were rinsed three times with PBS. Finally samples were mounted
with ProLong anti-fade mounting medium and imaged either with by
CFM (LSM 510 Meta NLO equipped with Argon, red HeNe, and
green HeNe lasers, and a Coherent Chameleon Ti-Sapphire laser; Carl
Zeiss, Thornwood, NY) or using a GE DeltaVision OMX system (GE
Healthcare Bio-Sciences, Pittsburgh, PA) for 3D-SIM.
For SV size measurements, the ImageJ measurement analysis tool
was used, and SV were measured at their Feret’s diameter, which is
the longest distance between any two points along the selection
boundary (45). For each condition, 3– 4 fields were evaluated, with
20 –30 SV per field measured from n ⫽ 3 mice.
Production and amplification of Ad expression constructs. The
recombinant replication-defective Ad expressing mouse CTSS-green
fluorescent protein (GFP) (Ad CTSS-GFP) was developed to inducibly express a COOH-terminal GFP-tagged CTSS under 1 ␮g/ml
doxycycline. The plasmid encoding full-length CTSS, prepro-CTSS,
was purchased from Open Biosystems (ORF length: 1,023 bp, Genbank accession number BC002125), and PCR was amplified with the
sense primer, 5=-CGCTAGCATGAGGGCTCCTGGCCAC-3=, and
the anti-sense primer, 5=-CGGTACCGCGATTTCTGGGTAAGAGCAATAACTAGC-3=. The PCR product was inserted into a pCR
II-TOPO vector from Invitrogen (Carlsbad, CA). The pCR II-TOPO
CTSS was then digested with restriction endonucleases, NheI and
KpnI, at the 5= and 3= sites of the pCR II-TOPO CTSS vector,
respectively. The resultant fragment encoding CTSS was then sub-
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RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
RESULTS
Fig. 1. Changes in tear protein secretion in male nonobese diabetic (NOD)
mice. The cathepsin S (CTSS) and ␤-hexosaminidase (␤-hex) activities were
measured as relative fluorescence units per microgram of tear protein. Total
tear protein concentration was measured as microgram of protein per microliter
of tear fluid. Data are presented as relative values to those from BALB/c mice,
which were arbitrarily set as 100. A: CTSS secretion, as determined by its
increased specific activity, was significantly higher in NOD mice; n ⫽ 8. B: no
significant change was detected in ␤-hex secretion from NOD mice; n ⫽ 9. C:
total protein level was significantly decreased in NOD mice; n ⫽ 9. *Significantly increased; #significantly decreased.
cultured for 18 h after replacement of fresh medium. The transduction
efficiency of each individual construct was ⬎80%. Cells were preincubated for 1 h, and then 100 ␮M CCh was applied to the treatment
group for 15 and 30 min to stimulate secretion. CTSS and ␤-hex
secretions were measured using the methods described above. After
the activity values were normalized to cell protein, the activity
increases were calculated by subtracting activities in medium of
preincubation groups from those of treatment groups.
CFM for live cell imaging. LGAC seeded in 35-mm glass-bottomed
Petri dishes were imaged in a 37°C incubation chamber. For analyzing
the distribution of CTSS-GFP, 70 nM LysoTracker Red DND-99 was
added to the medium 10 min before imaging. Imaging at shorter gene
expression times was ⬃12 h after transduction, whereas imaging at
longer gene expression times and cell videos was ⬃20 h after
transduction. For image capture of CTSS-GFP secretion in live cells,
images from a single confocal plane were taken sequentially for 80
cycles at a fixed time interval of 2.5 s. After the resting stage video,
CCh (100 ␮M) was added to the medium to stimulate exocytosis of
SV. When analyzing the colocalization of CTSS-GFP and YFPRab27b, the Emission Fingerprinting program was used to acquire a
complete lambda stack, followed by linear unmixing to distinguish the
close spectral fluorescence emission values of GFP and YFP.
Laser capture microdissection. LG were retrieved from NOD and
BALB/c mice, embedded in O.C.T. compound, and rapidly frozen
with liquid nitrogen removed under RNase-free conditions. The frozen blocks were cryosectioned and collected with membrane-coated
slides (PEN; Leica Microsystems, Buffalo Grove, IL) and stained with
hematoxylin. Acinar cells were then cut and collected by laser
microdissection systems (LMD7000; Leica Microsystems) following
the manufacturer’s protocol.
CTSS but not ␤-hex activity is increased in tears of NOD
mice with established disease. Male NOD mice as well as the
control strain, BALB/c, were analyzed at 12 wk, a time at
which significant lymphocytic infiltration of the LG is reliably
detected in male NOD mice (23, 51). Enzyme activity assays
were conducted with freshly collected tears from each mouse
strain. Compared with tears from BALB/c mice, tears from
NOD mice demonstrated a distinctive increase in the activity
level of CTSS (500%) as previously shown (28), but not in the
activity of another lysosomal enzyme normally found in tears,
␤-hex (46, 48) (Fig. 1). The total protein content in tears of
NOD mice was decreased by 33% (Fig. 1). Comparison of the
gene expression of these proteins in LGAC from each strain
using LCM and qPCR is shown in Table 1; mRNA for Ctss
was increased in LGAC of NOD mice, but Hexa, the ␣-subunit
of ␤-hex, was unchanged.
Rab3D gene expression level and distribution are altered in
NOD mouse LGAC. Previous data suggesting approximately
equivalent Rab3D protein levels in NOD and BALB/c mice
were obtained from whole LG lysates, which is not as reliable
as an indicator because of the large numbers of lymphocytes
present in the NOD sample (8). To investigate whether the
change in CTSS secretion from male NOD mouse LGAC was
associated with alterations in Rab protein expression and distribution, we explored the relevant Rab gene expression levels
Table 1. Gene expression data in NOD mice LGAC
compared with BALB/c
Strain
Gene
RQ
P Value
Change
NOD
Rab3d
Rab27a
Rab27b
Ctss
Hexa
0.6994
0.9269
0.8856
9.732
1.416
0.0006
0.307
0.1218
0.0006
0.2861
Decrease
None
None
Increase
None
NOD, nonobese diabetic; LGAC, lacrimal gland acinar cells; RQ, relative
quantity.
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Gene expression assays. RNA was prepared from whole LG from
C57 and all KO strains and from isolated acinar cells obtained by laser
capture microdissection (LCM) of NOD and BALB/c mouse LG,
using the RNeasy Plus Mini Kit and RNeasy Plus Micro kits, respectively. Reverse-transcription reactions were performed using TaqMan
reverse-transcription reagents to obtain cDNA from RNA. Quantitative PCR was conducted using an ABI 7900HT Fast Real-Time PCR
System. RT product (1 ␮l) (diluted with 8 ␮l of nuclease-free H2O),
1 ␮l of the primer, and 10 ␮l of universal master mix were used in
each reaction in a total volume of 20 ␮l. Succinate dehydrogenase
complex, subunit A was run as the internal control. The reaction
conditions and calculation methods were as described previously (51).
The recorded data were analyzed using the ⌬⌬Ct study calculating
function of the ABI software SDS 2.1. The relative quantity (RQ)
for a specific mRNA was obtained by calculations using the
equations ⌬Ct ⫽ Ct (studied mRNA) ⫺ Ct (housekeeping gene
mRNA), ⌬⌬Ct ⫽ ⌬Ct (mutation strain) ⫺ ⌬Ct (control strain), and
RQ (mutation strain/control strain) ⫽ 2⫺⌬⌬Ct.
Statistics. Data analysis was conducted to compare between sets
using either Student’s unpaired two-tailed t-test (for comparison of
NOD and BALB/c) or a one-way ANOVA followed by post hoc
analysis using Tukey’s test (for comparison between KO strains and
C57) as appropriate. The criterion for significance was at least P ⬍
0.05.
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RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
Fig. 2. Altered Rab3D distribution in male NOD mouse lacrimal gland acinar
cells (LGAC). Immunofluorescence was performed with lacrimal gland from
male BALB/c and NOD mice using anti-Rab3D or anti-Rab27b antibody. A
and B: Rab27b distribution was not significantly affected in NOD relative to
BALB/c mouse LGAC. C and D: abnormal basolateral distribution and an
apparent decrease in subapical accumulation of Rab3D-enriched vesicles was
detected in NOD compared with BALB/c mice (arrows). Z-stacks (E and F)
were taken to show the Rab3D distribution in BALB/c and NOD mouse
LGAC, respectively, throughout the sections. 4 BALB/c and 4 NOD mice were
used. *Lumen.
CTSS in tears from 3DKO mice is consistent with a possible
dysregulation of Rab27 isoform function in LGAC exocytosis
when Rab3D is absent and/or a negative regulation by Rab3D
of CTSS inclusion into secretory cargo. The trends of increased
activity of released CTSS and decreased total protein amount
in tears of NOD and 3DKO mice may be both linked to a
shared deficit in Rab3D function. From this point forward, we
compared the activities of secreted CTSS, a lysosomal tear
enzyme and putative disease biomarker exhibiting strong Rab
dependence for secretion, relative to ␤-hex, another lysosomal
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selectively in LGAC of NOD relative to BALB/c mouse. By
analyzing cDNA from LGAC obtained by LCM from NOD
mice, qPCR studies demonstrated that Rab3d expression was
significantly lower compared with BALB/c mice, whereas the
mRNA levels for Rab27a and Rab27b remained unchanged
(Table 1). Protein expression could not be assayed from LGAC
because of the limited amounts of material obtained from
acinar cells by LCM.
The distribution of Rab3D was also markedly changed in
NOD mice LGAC. Rab3D-enriched vesicles were detected in
the basolateral region, compared with their largely subapical
localization beneath the subapical actin lining the lumina in
BALB/c mice (Fig. 2, C–F; Supplemental Videos S1 and S2;
supplemental material for this article is available online at the
Journal website). In contrast, the distribution of Rab27b did not
change markedly (Fig. 2, A and B). These results suggested that
low Rab3D levels as well as Rab3D mislocalization may occur
in parallel with altered secretion of CTSS in NOD mouse LG,
prompting us to explore a possible link between these events.
CTSS secretion into CCh-stimulated tears is impaired in
mice lacking Rab27 isoforms but enhanced in mice lacking
Rab3D. We have previously shown that the cytoplasmic domain of the pIgR specifically associates with Rab3D and that
this association promotes recruitment of a population of pIgR
into Rab3D-enriched SV, presumably for enhanced release of
free SC, which is abundant in tears (13, 52). To better understand the association between Rab expression and enrichment
on SV and to expand our understanding of other cargoes
beyond pIgR, specifically CTSS and other lysosomal proteins
secreted to tears that may be selectively associated with specific Rabs and/or their adapter proteins, we utilized different
Rab KO strains, ashen, 27bKO, 27KO, 3DKO, and their parent
strain, C57 mice. As expected, the absence of Rab3D decreased release of SC into tears from 3DKO mice, further
confirming the association of a secretory population of pIgR
with Rab3D (Fig. 3). On the other hand, SC release in 27KO
mice was slightly but significantly increased.
In evaluating the tear proteins considered in Fig. 1 in these
KO mice, we found that CTSS activity was increased by 70%
in tears from 3DKO mice, but, remarkably, was decreased by
60 – 85% in tears from 27KO, ashen, and 27bKO mice relative
to its levels in tears from C57 mice (Fig. 4A). In contrast, ␤-hex
showed no significant changes in activity in tears from any of
the KO strains (Fig. 4B). The total tear protein content was
significantly decreased in 3DKO mice tears but increased in
tears from ashen and 27bKO mice (Fig. 4C).
These changes suggest an unanticipated complexity of sorting of lysosomal tear proteins in LGAC, as indicated by the
differential changes in tear protein abundance in response to
loss of specific Rab proteins. These findings suggest that some
proteins such as ␤-hex might proceed by constitutive trafficking into all mature SV and perhaps even into other constitutive
post-Golgi vesicles destined for the apical membrane, such that
impairment of individual mechanisms never significantly affects the total amount released. However, these findings suggest that other cargoes such as CTSS might require one or the
other Rab for selective recruitment or retention into a specific
SV population. Specifically, the observation that CTSS activity
is so significantly decreased in tears of mice lacking Rab27
isoforms suggests that its release into tears requires these
isoforms in some capacity; conversely, the increased release of
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
C947
CTSS-GFP and Ad YFP-Rab27b (7), our data indicated that,
when expressed for 20 h, CTSS-GFP was observed in Rab27benriched SV (Fig. 6B). Furthermore, these CTSS-GFP-bearing
vesicles were detected fusing at the apical plasma membrane
with release of fluorescent CTSS when LGAC were stimulated
by CCh (Supplemental Videos S3 and S4).
Analysis of immunofluorescence associated with the HEXA
subunit of ␤-hex showed that ␤-hex exhibited some colocalization with CTSS-GFP after 12 h of its expression (Fig. 7A,
arrows), when CTSS is present largely in lysosomes (Fig. 6A).
However, by 20 h of expression of CTSS-GFP, at which time
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Fig. 3. Changes in secretory component secretion in Rab3D knockout (3DKO)
and 27KO mice. The secretory component levels in tears from C57, 3DKO,
and 27KO were measured using Western blotting. Secretory component
release was significantly decreased in 3DKO and increased in 27KO. Secretory
component content was quantified as integrated intensity; n ⫽ 3 for each strain.
*Significantly increased; #significantly decreased.
tear enzyme with no strong Rab dependence for secretion, to
understand additional features of tear protein sorting in LGAC.
CTSS secretion is decreased from rabbit LGAC transduced
with Ad-encoding DN Rab27b. To further validate our hypothesis that CTSS secretion is influenced by the relative activity of
Rab3D and Rab27b, we transduced primary rabbit LGAC with
WT, DN, and CA Rab3D or Rab27b constructs. After CCh
stimulation, CTSS and ␤-hex secretion was measured. Total
and stimulated secreted CTSS activity was significantly decreased by 34 and 44%, respectively, when DN Rab27b was
expressed (Fig. 5A), consistent with our finding of reduced
CTSS secretion in 27bKO and 27KO mouse tears. We did not
detect any significant change in the activity of secreted CTSS
with expression of DN Rab3D relative to WT protein (Fig. 5B).
Moreover, overexpression of CA variants did not alter the
activity of secreted CTSS, suggesting that the Rab effectors,
rather than the Rab proteins themselves, may be the ratelimiting factors in this process. ␤-hex secretion also did not
change with expression of DN or CA forms of either Rab27b
or Rab3D (Fig. 5, C and D), consistent with in vivo data
(Fig. 4).
Exogenously expressed CTSS is secreted through Rab27benriched SV with CCh stimulation. As described above, increased CTSS activity in tears of NOD and 3DKO mice
suggests the increased secretion of this protein, prompting us to
hypothesize that its release may be accelerated in a compensatory manner mediated by Rab27 isoforms. To test this
hypothesis, we developed the Ad CTSS-GFP construct. When
expressed in simple cultured cells, this expressed protein is
localized to lysosomes, similar to the endogenous protein (data
not shown). As expected, when rabbit LGAC were transduced
for 12 h with Ad CTSS-GFP, CTSS-GFP was detected and
colocalized with lysosomes, as detected by LysoTracker Red
DND-99, a specific marker for acidic organelles such as late
endosome/lysosomes. However, when expression occurred for
a longer period of 20 h after transduction, the CTSS-GFP
signal was largely redistributed concentrated around the lumen
(Fig. 6A). By analyzing rabbit LGAC transduced with both Ad
Fig. 4. Changes in tear protein secretion in Rab KO mice. The CTSS and ␤-hex
activities were measured as relative fluorescence units per microgram of tear
protein. Total tear protein concentration was measured as micrograms of
protein per microliter of tear fluid. Values are presented as relative values
relative to those from C57 mice, which were arbitrarily set as 100. A: CTSS
secretion was significantly higher in 3DKO mice (n ⫽ 10) but lower in 27KO
(n ⫽ 12), ashen (ash) (n ⫽ 6) and 27bKO (n ⫽ 8) mice compared with C57
mice (n ⫽ 29). B: ␤-hex secretion was not significantly altered in tears from
any of the KO strains; n ⫽ 61 for C57, n ⫽ 13 for 3DKO, n ⫽ 33 for 27KO,
n ⫽ 21 for ashen, n ⫽ 8 for 27bKO. C: total protein content was significantly
decreased in 3DKO mice (n ⫽ 36), whereas its level was significantly
increased in ashen (n ⫽ 8) and 27bKO (n ⫽ 7) mice; n ⫽ 35 for C57, n ⫽ 18
for 27KO. *Significantly increased; #significantly decreased.
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RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
it becomes enriched beneath the apical plasma membranes and
within SV (Fig. 6), ␤-hex still retains a relatively broad
distribution with little colocalization with CTSS-GFP (Fig.
7B). Although ␤-hex immunofluorescence is abundant
throughout the cell, it does not appear to be present in a
polarized distribution enriched beneath apical plasma membranes. Evaluation of its enrichment with SV labeled with
YFP-Rab27b (Fig. 7C) and mCherry-Rab3D (Fig. 7D) in the
subapical region showed traces of colocalization (arrows) but
not a striking enrichment of total cellular protein within these
organelles. This distribution suggests that its presence in tears
may be attributable to its abundance in a number of vesicle
types mobilized in response to CCh stimulation, consistent
with the lack of a specific effect of Rab3D or Rab27 KO on its
secretion into tears (Fig. 4).
KO of one Rab does not affect the distribution or stimulation-induced reorganization of the other. Previous studies have
suggested colocalization of Rab3D and Rab27b isoforms on
the same SV in LGAC (7), whereas Rab3A and Rab27a
isoforms are likewise colocalized on SV in neurons (19).
Various roles have been suggested for these isoforms in exocytosis. So far, we have suggested a functional distinction for
Rab3D and Rab27 isoforms in association with particular
cargoes by showing pIgR association with Rab3D and CTSS
association with Rab27b.
To further elucidate the relationship between these Rab
isoforms, we determined whether the absence of one Rab
resulted in a compensatory upregulation of the gene expression
of the other Rab proteins, measuring gene expression using
qPCR. Our data showed that mRNA expression of Rab3d in
27KO and Rab27a and Rab27b in 3DKO remained unchanged
(Table 2). Intriguingly, the expression of Ctss and Hexa genes
were significantly decreased in 27KO LG while being significantly increased in 3DKO LG (Table 2). The increase in gene
expression in 3DKO mouse LG of CTSS was paralleled by its
significant increase in activity in 3DKO LG lysates to 230% of
control, while CTSS activity level in 27KO and ␤-hex activity
in 27KO and 3DKO were unaffected (Table 3). The increased
gene expression in 3DKO mouse LG in Hexa was not associated with increased ␤-hex activity. This finding suggests that
increased cellular CTSS may follow reductions in Rab3D
isoform expression.
To determine whether both Rabs are localized to SV that
respond to M3 receptor activation and thereby participate in the
regulated secretory pathway, one LG of the anesthetized mouse
was stimulated by CCh, and then the unstimulated (resting) LG
and CCh-stimulated LG from the same mouse were isolated
and processed immediately after the mouse was euthanized for
detection of Rab proteins by immunofluorescence. In the C57
mouse LGAC, both Rab3D and Rab27b were associated with
large apparent SV (⬃1 ␮m in diameter) clustered closely to the
apical membrane, which were easily localized by their proximity to the abundant subapical actin network (Fig. 8). In
stimulated LG, both Rab3D and Rab27b immunofluorescence
was associated with irregularly enlarged structures (2-fold
increase in diameter), consistent with previous studies showing
that SV may form compound fusion intermediates in stimulated rabbit LGAC in vitro (25, 29). However, this is the first
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Fig. 5. CTSS and ␤-hex secretion from rabbit LGAC transduced with wild-type (WT), dominant negative (DN), and constitutively active (CA) Rab27b or Rab3D
constructs. CTSS secretion from rabbit LGAC transduced with WT, DN, and CA Rab27b (A) or Rab3D (B) constructs and ␤-hex secretion from rabbit LGAC
transduced with WT, DN, and CA Rab27b (C) or Rab3D (D) constructs is shown. CTSS and ␤-hex secretion into culture medium is shown as enzyme activity
normalized to cell pellet protein. All values were expressed as a percentage of control (basal, WT) samples. The stimulated increment was calculated by
subtracting values obtained from basal secretion (basal) from values obtained from total secretion (total). Assays were repeated 7 times (A and C) and 5 times
(B and D) with triplicate measurements for each data point in each assay. *Significantly decreased.
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
C949
time this has been shown in mice in situ. SV morphological
changes consistent with exocytosis were observed in both C57
and KO strains (Fig. 8), suggesting that SV containing either
Rab3D or Rab27b are fusion competent in the absence of the
other.
Rab3D and Rab27 reside in distinct domains on SV. To
characterize further any overlap in the function of these distinct
Rab proteins in regulated exocytosis in LGAC, we analyzed
the extent of colocalization of Rab3D and Rab27 on SV. First,
we utilized primary rabbit LGAC for CFM analysis of exogenous fluorophore-tagged Rab27b. Primary LGAC were transduced with Ad YFP-Rab27b. Fixed cells were then immunolabeled with primary and fluorescent secondary antibodies to
detect Rab3D or Rab27a. YFP-Rab27b was expressed on large
vesicles beneath the apical membrane and showed a high
extent of colocalization with both Rab3D and Rab27a (Fig.
9A). Analysis of immunofluorescence was also performed with
mouse LG tissue sections. Because the anti-Rab3D and antiRab27b antibodies are both from rabbit, we were not able to
perform dual labeling with Rab3D and Rab27b. However, in
regions of high Rab27a enrichment, this protein was highly
colocalized both with Rab3D and Rab27b in acinar cells within
C57 mouse LG (Fig. 9B). The basolateral membrane labeling
of Rab27a is due to nonspecific secondary antibody binding by
the plasma membrane because the Rab27a antibody is of
mouse source and the anti-mouse secondary antibody reacts
with residual immunoglobulins in the interstitium and at the
plasma membrane.
To further reveal the details of Rab distribution and colocalization, particularly on the most apical SV, that could be
limited by the resolution achievable by CFM, we utilized
3D-SIM to investigate the Rab localization on individual SV in
mouse LG sections (Fig. 9C). 3D-SIM images of SV enriched
in Rab27a and Rab3D or Rab27a and Rab27b in BALB/c mice
LG revealed discrete microdomains of both Rabs on single SV
membranes that were enriched in one of the other Rab isoforms. These data demonstrate that Rab3D and Rab27 isoforms
reside on the same SV in LGAC but may occupy distinct
microdomains and be present in different abundance across
SV, consistent with the possibility that they may fulfill different functions during the maturation of the SV, including the
recruitment of different cargo proteins or the retrieval of other
cargoes from SV to endosomes or the trans-Golgi network.
Customization of the extent of the individual Rabs on any
given SV may thus significantly affect their content proteins
and, consequently, the composition of the tear film.
DISCUSSION
We found an increase in CTSS gene expression in LGAC
and its increased enzyme activity in tears from 3DKO mice,
similar to findings in NOD mouse (28) and patients with SS
(18), leading to the hypothesis that a loss of general Rab3D
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Fig. 6. CTSS-green fluorescent protein (GFP) distribution in rabbit primary LGAC. A: CTSS-GFP
was largely colocalized with LysoTracker Red
DND-99 after transduction with adenoviral (Ad)CTSS-GFP in LGAC for 12 h; however, after 20 h,
CTSS-GFP was largely relocated from lysosomes to
the subapical region although trace amounts of fluorescence could still be detected in lysosomes. B:
Ad-mediated overexpression of CTSS-GFP cotransduced with Ad-yellow fluorescent protein(YFP)Rab27b showed CTSS-GFP recovery in a subset of
Rab27b-enriched secretory vesicles (arrows) at 20 h.
*Lumen.
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RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
Table 2. Gene expression data in Rab knockout mice LG
compared with C57
Strain
Gene
RQ
P Value
Change
3DKO
Rab27a
Rab27b
Ctss
Hexa
Rab3d
Ctss
Hexa
0.8015
0.8175
2.879
1.312
1.066
0.3994
0.7588
0.0924
0.2336
0.0047
0.0005
0.5216
0.0019
0.0002
None
None
Increase
Increase
None
Decrease
Decrease
27KO
3DKO, Rab3D knockout.
function may contribute to the altered profile of tear protein
secretion from LGAC in mouse models of and patients with
SS. This hypothesis is supported by our findings of lower gene
expression level and altered Rab3D localization in NOD
LGAC; moreover, this is consistent with recent studies showing that Rab3D localization is altered in LGAC from patients
with SS compared with normal controls (26). A similar alteration in Rab3D localization combined with decreased Rab3D
Table 3. CTSS and ␤-hex activity (relative fluorescence unit
per microgram protein) in Rab KO mice LG relative to C57
Strain
Protein
% of C57
P Value
Change
3DKO
CTSS
␤-hex
CTSS
␤-hex
230
111.3
60
108.3
0.0074
0.4036
0.1107
0.4699
Increase
None
None
None
27KO
CTSS, cathepsin S; ␤-hex, ␤-hexosaminidase.
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Fig. 7. ␤-hex distribution in rabbit primary LGAC. HEXA immunostaining to
detect ␤-hex was performed in rabbit LGAC transduced with Ad CTSS-GFP,
12 h (A); CTSS-GFP, 20 h (B); YFP-Rab27b (C); and mCherry-Rab3D (D)
constructs. ␤-hex showed traces of colocalization with CTSS-GFP at 12 h of
expression in subapical and basolateral regions (A), and with YFP-Rab27b (C)
and mCherry-Rab3D (D) in the subapical region (arrows). No colocalization
with CTSS-GFP at 20 h of expression was detected (B). Regions in white
boxes were shown in higher magnification with separate channels. Arrows,
colocalization. *Lumen.
protein levels in salivary gland acinar cells of patients with SS
was also demonstrated in a separate study (2). Because Rab3D
and Rab27 are associated with regulated tear protein exocytosis
(7, 13, 52), we focused in the remainder of this study on
distinguishing the roles of these two Rab subfamilies in regulating the secretion of CTSS from LGAC.
Our data suggest that CTSS secretion from LGAC is mediated by Rab27 isoforms. This hypothesis is supported by our
findings that 1) CTSS activity in tears is markedly reduced
when either Rab27 isoform is lacking (Fig. 4); 2) CCh-stimulated recovery of CTSS activity into medium is significantly
decreased from LGAC transduced with DN-Rab27b (Fig. 5);
and 3) CTSS-GFP is detected in and secreted from YFPRab27b-enriched SV in rabbit LGAC, suggesting sorting of
CTSS into these SV (Fig. 6B, Supplemental Videos S3 and
S4). Surprisingly, the activity of another lysosomal enzyme,
␤-hex, found in tears, is not increased under these conditions,
suggesting that it is not mediated by Rab27 isoforms.
Previous work has shown that endogenous Rab3D and
Rab27b exhibit significant colocalization by immunostaining
(7), verified in our studies reported here by CFM. The high
resolution provided by 3D-SIM superresolution microscopy
provided further insights regarding Rab distribution on individual SV, revealing that some Rab3D and Rab27 are colocalized, whereas some are enriched in separate microdomains on
the same SV. Previous studies have shown localization of
different Rab proteins on distinct domains of other organelles.
Rab4, Rab5, and Rab11 label distinct domains on the same
early and recycling endosomal membranes in A431 cells (42).
Rab7 and Rab9 occupy distinct membrane domains on late
endosomes in BSC-1 cells (3). Rab proteins also regulate
membrane trafficking by recruiting effector proteins that select
cargoes by interaction with specific elements of cargo proteins.
Examples include the interaction of TIP47, a Rab9 effector,
with the cytoplasmic domain of mannose 6-phosphate receptor
(M6PR) and retromer, a Rab7 effector, with its cargo proteins
(40). When CTSS is targeted to lysosomes, it is modified with
M6P residues (50), whereas other enzymes such as cathepsin D
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
C951
can be transported to the lysosomes through an additional
sortilin-mediated pathway. By extension from these studies, we
suggest that different populations of SV containing different
content proteins distinguished by varying abundances of
Rab3D or Rab27 isoforms exist in LGAC (Fig. 10), with the
different Rab isoforms participating in tailoring of the content
of these SV through interaction with unknown effectors. This
balance of Rab isoforms is affected in SS, promoting the
development of SV depleted in Rab3D and enriched in Rab27
isoforms and, as a result, containing more CTSS. The future
investigations of interactions between these Rabs and their
effector proteins to mediate or recruit cargo proteins may help
us to explain this apparent modulation of cargo secretion by
these SV Rabs.
Unlike CTSS, changes in expression of Rab3D or Rab27
isoforms do not impact the regulated secretion of ␤-hex into
tears (Figs. 4 and 5). Certainly, the distribution of ␤-hex
immunofluorescence does not suggest relative enrichment of
this protein in Rab3D and/or Rab27-enriched SV vs. other
cellular membranes (Fig. 7). This apparent difference in the
post-Golgi trafficking of these two lysosomal proteins, CTSS
and ␤-hex, into tears in Rab KO mouse models is consistent
with our recent findings on their disparate secretion into tears
of pearl mouse. The pearl mouse has a mutation in the Ap3b1
gene, which encodes the ␤3A subunit of the AP-3 adaptor,
which is involved in protein transport from the trans-Golgi
network to lysosomes. Both CTSS and ␤-hex are thought to
interact similarly with M6PR in the trans-Golgi network to be
actively recruited to lysosomes (27, 50), suggesting similar
consequences to their sorting associated with reduced AP-3mediated transport. However, in pearl mice, ␤-hex was increased by twofold into tears, as verified both by enzyme
activity assay and comparative tear proteomics (54), whereas
CTSS secretion into pearl mouse tears is completely unaffected (Fig. 11). Our interpretation of this difference is that
␤-hex proceeds by default to one or more post-Golgi compartments in LGAC that can be mobilized by secretagogues, in the
absence of its normal recruitment to lysosomes from the
trans-Golgi network caused by the pearl mutation. The observation that CTSS is not comparably increased in pearl mouse
tears suggests, in LGAC, that another process may exist to
actively mediate selective CTSS sorting, possibly including
Rab27 proteins and their effectors. Alternatively, a cellular
pool of ␤-hex lacking M6P may exist, creating differences in
the post-Golgi trafficking between these two pools that does
not exist for CTSS.
Rab27a has long been identified as a regulator for the
transport or exocytosis of lysosome-related organelles (LRO)
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Fig. 8. Rab3D and Rab27b distribution in C57 and Rab
KO mice LGAC under resting conditions or after in situ
topical stimulation with carbachol. Immunofluorescence
was performed with LG sections using anti-Rab3D or
anti-Rab27b antibody. Both Rab3D (A) and Rab27b (B)
were localized to vesicular structures of large diameter,
consistent with mature secretory vesicles. In stimulated
C57 LG, both Rab3D- and Rab27b-enriched secretory
vesicles showed irregularly enlarged structures (arrows)
compared with acini from unstimulated or “resting” LG,
suggestive of the multivesicular fusion intermediates
identified previously. Comparable distribution and reorganization of secretory vesicles was detected in KO
strains with the labeling of the remaining Rab, relative
to C57 mice. Resting and stimulated LG analyzed in
parallel were from the same mouse. *Lumen. Quantification of the Feret’s diameter of secretory vesicles in
each strain under each condition is shown to the right of
the images. In graph, *significantly increased.
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RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
such as melanosomes in melanocytes (22), lytic granules in
cytotoxic T lymphocytes (43), and dense granules in platelets
and secretory granules in mast cells (33). Because LROs are
primarily secretory organelles carrying lysosomal cargo proteins, Rab27-enriched SV in LGAC may have some properties
of LRO. Some evidence of the direct trafficking of lysosomal
proteins like CTSS directly from lysosomes to an LRO-like SV
includes 1) demonstration that LGAC from 27bKO and 27KO
mice exhibits increased numbers of lysosomes in parallel with
decreased SV (7) and 2) findings that overexpressed CTSSGFP first becomes enriched in lysosomes and then is later
localized to Rab27b-enriched SV (Fig. 6). Although the processes governing release of CTSS from LGAC certainly imFig. 9. Distribution of Rabs in rabbit primary LGAC by confocal fluorescence
microscopy (A), C57 mouse LG by confocal fluorescence microscopy (B), and
BALB/c mouse LG (C) by 3D-structured illumination microscopy. A: rabbit
LGAC transduced with Ad YFP-Rab27b were immunolabeled with antiRab3D or anti-Rab27a antibody. YFP-Rab27b was colocalized to secretory
vesicles in the subapical region enriched with Rab3D and Rab27a. This was
repeated with 3 cell preparations. B: C57 mouse LG sections were immunolabeled to detect Rab3D and Rab27 isoforms and imaged by confocal fluorescence microscope. Rab27a was similarly colocalized with Rab3D and Rab27b.
The plasma membrane staining observed for Rab27a was due to the secondary
anti-mouse antibody reactions with residual immunoglobulins in the interstitium and at the plasma membrane; 3 mice were analyzed. C: BALB/c mouse
LG sections were immunolabeled and imaged by 3D-structured illumination
microscope and revealed domains on the same secretory vesicles enriched in
Rab27a (empty arrows) and Rab3D (solid arrow)/Rab27b (arrowhead); 3 mice
were analyzed. *Lumen.
Fig. 11. Secretion of CTSS in pearl mouse tears. CTSS activity was measured
as relative fluorescence units per microgram of tear protein. Data are presented
as relative values to those from C57 mice, which were arbitrarily set as 100.
CTSS secretion was unchanged in pearl mouse tears; n ⫽ 14 for C57 and 15
for pearl mice.
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Fig. 10. Working model for functions of Rab3D and Rab27 in exocytosis of
tear proteins in healthy and Sjögren’s Syndrome (SS) LGAC. Rab3D and
Rab27b are located to secretory vesicles but with different abundances.
Whereas some content proteins may reach secretory vesicles and other organelles from the trans-Golgi through constitutive trafficking, such as ␤-hex, other
content proteins may be selectively recruited into these secretory vesicles
through effectors associated either with Rab3D (e.g., polymeric immunoglobulin receptor) or Rab27 (e.g., CTSS), either at the trans-Golgi network (TGN)
or by trafficking from other membrane compartments. We speculate that the
recruitment of low amounts of CTSS to secretory vesicles normally occurs
from endo-lysosomal compartments in a process driven by one or both Rab27
isoforms, which then remain associated with secretory vesicles enriched
largely in Rab3D beneath the apical plasma membrane. In SS, Rab3D gene and
protein expression are decreased, and the distribution of some cellular Rab3D
protein is shifted to accumulation with large basolateral organelles. The loss of
Rab3D on secretory vesicles may result in decreased recruitment of some tear
proteins to secretory vesicles and also permits and/or promotes the increased
flow of CTSS through Rab27-mediated processes to generate altered secretory
vesicles with a relative enrichment in Rab27 isoforms containing increased
CTSS. LYS, lysosome; CS, secretory component.
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME
ACKNOWLEDGMENTS
We thank Dr. Dietmar Riedel (Max Planck Institute for Biophysical
Chemistry) for the generous gift of the 3DKO mouse breeding pairs. We thank
Dr. Serhan Karvar (University of South Carolina) for the kind gift of the Ad
YFP-Rab27b constructs. We thank Francie Yarber for primary rabbit LGAC
preparations and adenoviral construct purification, Srikanth Janga for assistance with mouse tear collection and analysis of data, and Chuanqing Ding for
critical comments during manuscript preparation. We also acknowledge the
support of the Cell & Tissue Imaging Core at the Research Center for Liver
Disease, University of Southern California for their laser capture microscopy
services, and Marc Green and the Center for Electron Microscopy and
Microanalysis (CEMMA) at the University of Southern California for 3D-SIM
services.
GRANTS
This work was supported by NIH R01 EY011386 to (S. Hamm-Alvarez)
and by an unrestricted departmental grant from Research to Prevent Blindness
(RPB), New York, NY 10022. The Cell & Tissue Imaging Core at the
Research Center for Liver Disease, University of Southern California is
supported by NIH grant P30 DK048522.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Z.M., M.C.E., C.T.O., and S.F.H.-A. conception and design of research;
Z.M., P.-Y.H., C.-Y.C., W.K., and T.T. performed experiments; Z.M., M.C.E.,
and C.-Y.C. analyzed data; Z.M., M.C.E., and S.F.H.-A. interpreted results of
experiments; Z.M., M.C.E., and P.-Y.H. prepared figures; Z.M. drafted manuscript; Z.M., M.C.E., P.-Y.H., T.T., C.T.O., and S.F.H.-A. edited and revised
manuscript; Z.M. and S.F.H.-A. approved final version of manuscript.
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19.
RABS REGULATE TEAR CATHEPSIN S IN SJÖGREN’S SYNDROME