Human Molecular Genetics, 2015, Vol. 24, No. 11
3181–3191
doi: 10.1093/hmg/ddv070
Advance Access Publication Date: 20 February 2015
Original Article
ORIGINAL ARTICLE
Blocking hyperactive androgen receptor signaling
Jin-Song Shen1,*, Xing-Li Meng1, Mary Wight-Carter2, Taniqua S. Day1,
Sean C. Goetsch3, Sabrina Forni1, Jay W. Schneider3, Zhi-Ping Liu3
and Raphael Schiffmann1
1
Institute of Metabolic Disease, Baylor Research Institute, 3812 Elm Street, Dallas, TX 75226, USA, 2Animal
Resources Center Diagnostic Laboratory and 3Department of Internal Medicine, University of Texas Southwestern
Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
*To whom correspondence should be addressed. Tel: +1 2148204533; Fax: +1 2148204853; Email: [email protected]
Abstract
Fabry disease is caused by deficient activity of lysosomal enzyme α-galactosidase A. The enzyme deficiency results in
intracellular accumulation of glycosphingolipids, leading to a variety of clinical manifestations including hypertrophic
cardiomyopathy and renal insufficiency. The mechanism through which glycosphingolipid accumulation causes these
manifestations remains unclear. Current treatment, especially when initiated at later stage of the disease, does not produce
completely satisfactory results. Elucidation of the pathogenesis of Fabry disease is therefore crucial to developing new
treatments. We found increased activity of androgen receptor (AR) signaling in Fabry disease. We subsequently also found that
blockade of AR signaling either through castration or AR-antagonist prevented and reversed cardiac and kidney hypertrophic
phenotype in a mouse model of Fabry disease. Our findings implicate abnormal AR pathway in the pathogenesis of Fabry
disease and suggest blocking AR signaling as a novel therapeutic approach.
Introduction
Fabry disease is an X-linked lysosomal storage disorder. It is
caused by deficient activity of lysosomal enzyme α-galactosidase
A (α-gal A) (1). Deficiency of α-gal A results in an inability of the
cells to catabolize glycosphingolipids (GSL) with terminal α--galactosyl residues. These GSLs, particularly globotriaosylceramide
(Gb3), accumulate progressively in virtually all organs and systems, resulting in progressive multisystemic damage (2,3).
Fabry disease exhibits a variety of clinical manifestations, of
which cardiovascular and renal complications are the most life
threatening. Left ventricular hypertrophy is a key feature in
Fabry disease. In most cases, the hypertrophy is concentric, leading to diastolic dysfunction and fibrosis. Systolic dysfunction
occurs later in the course of the disease (4). Renal manifestations
occur in most Fabry patients. Kidneys of Fabry patients are enlarged in the third decade of life, followed by gradually decreasing
size after the fourth decade (5). Proteinuria and decreased glomerular filtration rate progress over time, leading to end-stage
renal disease (2,3). Although male patients usually have the
most severe form of the disease, heterozygous female patients
may also be affected, and disease severity depends also on the
degree of inactivation of the affected X-chromosome in each
organ (6).
Enzyme replacement therapy (ERT) is currently the only
specific treatment available for this disease. ERT is effective
in reducing GSL accumulation in tissues and appears to slow
Received: January 18, 2015. Revised and Accepted: February 16, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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ameliorates cardiac and renal hypertrophy in Fabry
mice
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Results
Phenotypes of Fabry mice
We found that both hemizygous male and homozygous female
Fabry mice develop late-onset cardiac hypertrophy. Compared
with wild-type (WT) controls with the same strain background,
hemizygous male Fabry mice had significantly increased heartto-body weight ratio at 12 and 18 months of age, but not at 5
months (Fig. 1A). Echocardiography showed significantly increased left ventricular (LV) wall thickness and LV mass in 18month-old male Fabry mice (Fig. 1B and C). There was no change
in LV diameters (diastolic, 3.1 ± 0.3 mm in WT, 3.1 ± 0.2 mm in
Fabry) and percent fractional shortening (53.4 ± 3.7% in WT,
55.3 ± 5.7% in Fabry) (n = 7). Expression levels of markers of cardiac hypertrophy, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and α- and β-myosin heavy chain (MHC) in
heart tissues were analyzed by quantitative RT–PCR. ANP was
significantly increased in male Fabry mice in an age-dependent
manner (Fig. 1D). There was no significant change in BNP and
α- and β-MHC (Supplementary Material, Fig. S1A–D). It is known
that activated Akt is associated with cardiac hypertrophy (15).
Akt activation requires the phosphorylation of the serine residue
at position 473 and of the threonine residue at position 308. We
found significantly increased phosphorylated Akt (Ser473) in
male Fabry mouse hearts compared with WT controls (Fig. 1E).
Parallel to the findings in heart mass and ANP, the increased phosphorylation of Akt was seen at 18 months, but not at 5 months of
age. Masson’s Trichrome staining revealed no interstitial fibrosis
Figure 1. Phenotypes of hemizygous male Fabry mice. (A) Heart-to-body weight ratio at various time points (n = 11–16). (B and C) LV wall thickness (B) and LV mass (C) of 18month-old mice measured by echocardiography (n = 7). (D) ANP mRNA levels in heart tissues measured by real-time RT–PCR (n = 5–9). (E) Phosphorylation of Akt (Ser473) in
mouse heart tissues. Upper: representative images of western blot in 18-month-old mouse heart; Lower: intensity of the bands analyzed by densitometry (n = 3). (F) Kidneyto-body weight ratio (n = 4–12). (G–I) Histological examination of 18-month-old mouse kidneys. Cortex width (G) and tubule thickness (H) were quantified using computerassisted image analysis (n = 3–4). (I) Increased proximal tubule wall thickness and increased number of vacuoles (arrows) in proximal tubules in Fabry mouse kidneys. Scale
bar, 50 μm. (J) Testicular weight of 12-month-old mice (n = 8). (K) Weight of subcutaneous white adipose tissue in 12-month-old mice (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001
determined by t-test.
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progression of the disease (7). Recent studies suggested that ERT
is more efficacious when initiated at earlier disease stages (8,9).
In general, however, the clinical effect of ERT in Fabry disease is
insufficient to stop or reverse most disease processes when initiated in adults. There is therefore significant need to develop
novel therapeutic approaches that will be based on extending
our understanding of Fabry disease beyond the primary enzyme
deficiency.
The mechanisms by which accumulation of GSL leads to the
clinicopathological manifestations of Fabry disease are poorly
understood. Previous studies suggested that oxidative stress
and endothelial nitric oxide synthase dysfunction are involved
in the vasculopahy of this disease (10,11). Recently, it has been
found that the level of deacylated Gb3 (lyso-Gb3) is markedly elevated in Fabry patients’ plasma and that lyso-Gb3 may contribute
to vascular remodeling through proliferation of smooth muscle
cells (12). In addition, the lysosomal-autophagy system was
found to be abnormal in Fabry disease (13). However, no precise
molecular mechanism for cardiac hypertrophy and renal manifestations has been proposed. The goal of our research was to
identify the responsible signaling pathway and modulate it for
therapeutic purposes.
In this study, we characterized the mouse model of Fabry disease (α-gal A knockout mouse) (14) and identified several previously unreported disease phenotypes. These phenotypes
provided a clue to abnormal androgen receptor (AR) signaling
and indicated the possibility of mitigating the disease manifestations in a mouse model of Fabry disease.
Human Molecular Genetics, 2015, Vol. 24, No. 11
Increased AR activity in Fabry mice and Fabry patients’
cells
We found that all the above abnormalities in Fabry mice can be
explained by excess androgen action. It is known that androgens
cause cardiac hypertrophy (16,17), as well as increased kidney
size (18,19). The histological basis of androgen-induced kidney
hypertrophy is hypertrophied proximal tubules (19). Androgens
may regulate testicular size, as suggested by the small testes in
AR-deficient mice (20). Androgens inhibit fat deposition by blocking a signal transduction pathway that normally supports adipocyte function (21). AR-deficient mice develop obesity and kidney
atrophy (22).
We therefore tested whether androgen action is enhanced in
Fabry mice. Androgens exert their biological roles mainly
through AR, which is a ligand-dependent transcription factor.
The binding of androgen to the AR results in translocation of
AR from the cytosol into the nucleus, where AR binds to androgen
response element (ARE) and regulates the expression of androgen-regulated genes (or AR-target genes) (23). Transcription levels
of androgen-regulated genes reflect the activity of AR signaling.
Insulin-like growth factor 1 (IGF-1) is a well-characterized androgen-regulated gene, which is positively regulated by AR (24). The
expression of IGF-1 was significantly upregulated in male Fabry
mouse heart (Fig. 2A). Transforming growth factor-β1 (TGF-β1),
which is negatively regulated by AR (25), was downregulated
in Fabry mouse heart (Fig. 2A). β-Glucuronidase (Gusb), kidney androgen-regulated protein (Kap) and acyl-CoA synthetase mediumchain family member 3 (Acsm3, also known as SA gene), the most
established androgen-regulated genes in mouse kidneys (19,26),
were all significantly upregulated in male Fabry mouse kidneys
(Fig. 2A). In addition to mRNA levels, Gusb enzyme activity in kidneys was significantly increased in Fabry mice (Fig. 2B). The Gusb
activity correlated well with its mRNA levels, suggesting that the
increased activity is caused by upregulated gene transcription
(Fig. 2C). Androgens increase urinary excretion of Gusb (18). Gusb
activity in urine was significantly increased in male Fabry mice
compared with WT controls (Fig. 2D). Collectively, these data
strongly suggested that the activity of AR signaling is increased
in male Fabry mouse hearts and kidneys.
We also tested whether AR signaling is abnormally activated
in homozygous female Fabry mice. Consistent with that of male
mice, cardiac expression of IGF-1 was significantly upregulated in
female Fabry mice compared with WT controls (Fig. 2E). However,
the regulation of androgen-regulated genes in female Fabry
mouse kidneys differed clearly from that of the male Fabry
mice. Gusb and Acsm3 mRNA levels were unchanged in homozygous female Fabry mouse kidneys relative to WT controls, and
Kap transcription was significantly downregulated in female
Fabry mice (Fig. 2F). Parallel to the mRNA level, kidney Gusb activity was unchanged in female Fabry mice.
The increased activity of AR signaling can be caused either by
increased levels of ligands or increased transcriptional function
of AR. We found blood testosterone levels to be similar in male
Fabry mice compared with WT controls at both 2 and 12 months
of age (Supplementary Material, Fig. S3A and B). This suggested
that enhanced AR signaling in Fabry mice may be caused by increased AR activity.
To assess transcriptional activity of AR, we performed an AR
reporter gene assay in primary cultured renal proximal tubules
isolated from Fabry mice. The proximal tubules isolated from
12 month-old male Fabry and WT mice were infected with adenovirus encoding luciferase gene controlled by an ARE-containing
promoter (Ad/MLUC7) (27). Dihydrotestosterone (DHT) could not
induce luciferase expression through endogenous AR in either
Fabry or WT-derived tubule cells (Fig. 2G), suggesting that AR expression level under this experimental condition is not sufficient
to transactivate the reporter expression. When exogenous AR
was co-expressed by Ad5hAR (27), DHT induced luciferase expression, and this AR-transactivation was moderately, but significantly, enhanced in Fabry mouse tubule cells compared
with WT (Fig. 2G).
To further determine whether the aberrant AR signaling is
also present in human patients, we tested Fabry patient-derived
microvascular endothelial cell line (IMFE1) (28). IMFE1 cells have
decreased α-gal A activity and accumulated Gb3. To obtain isogenic ‘normal’ control cells, a normal α-gal A (GLA) cDNA was retrovirally introduced into IMFE1 cells [designated as IMFE1(α-gal
+)]. α-Gal A deficiency was corrected and Gb3 accumulation was
cleared in IMFE1(α-gal+) cells compared with mock-treated
IMFE1 cells [IMFE1(mock)] (Fig. 2H and I). To compare gene profile
in this pair of diseased and ‘cured’ cells, we performed a PCR
array, which constitutes 84 endothelial cell biology-related
genes including prostate-specific antigen (PSA or KLK3), the
best known androgen-regulated gene in humans (29). There
were only two genes whose expressions significantly differed
(>3-fold difference) in IMFE1(α-gal+) and IMFE1(mock) cells, and
interestingly, one of these was PSA. PSA mRNA level in IMFE1
(mock) was 3.9-fold than that in IMFE1(α-gal+) cells (Fig. 2J). A
complete list of the examined genes and their expression levels
is included in Supplementary Material, Table S1.
Gb3 accumulation is associated with upregulated AR
transcription
We asked whether the increased AR activity in Fabry mouse tissues is caused by increased AR gene expression. AR protein level
was not increased in Fabry mouse hearts (Fig. 3A) and kidneys
(not shown), suggesting that the higher gene expression level
was not causal. Interestingly, however, we found increased AR
mRNA levels in male Fabry mouse hearts and kidneys at most
time points (Fig. 3B and C). AR mRNA in 12-month-old female
Fabry mouse hearts trended towards a higher level relative to
WT mice (Fig. 3D). AR mRNA level correlated well with expression
levels of androgen-regulated genes in mouse tissues (Supplementary Material, Fig. S4A–D), suggesting a potential link
between upregulated AR and the increased activity of AR signaling. To more directly determine the role of GSL on AR
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in either Fabry mice or WT at any analyzed time point. Homozygous female Fabry mice had similar age-dependent hypertrophic
findings (Supplementary Material, Fig. S2A–C).
We also found that both sexes of Fabry mice develop progressive renal hypertrophy. Male Fabry mice had remarkably
increased kidney-to-body weight ratio after 12 months of age
(Fig. 1F). Histological analysis showed increased width of the
renal cortex and thickness of the proximal tubules in 18month-old male Fabry mice (Fig. 1G and H). Fabry mice had a significantly increased number of intracellular vacuoles in proximal
tubule epithelial cells (Fig. 1I). Urine albumin/creatinine ratio and
plasma creatinine and BUN levels in male Fabry mice were similar to WT mice at 14–16 months. Homozygous female Fabry mice
had enlarged kidneys with the same time course as male Fabry
mice (Supplementary Material, Fig. S2D).
In addition, we found significantly increased testicular weight
in Fabry mice (Fig. 1J). Fat deposition was significantly decreased
in both male and female Fabry mice compared with WT controls
at 12 months (Fig. 1K and Supplementary Material, Fig. S2E).
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hemizygous male Fabry mice and WT controls measured by real-time RT–PCR (n = 6–7). (B) Gusb activities in kidneys from 18-month-old male Fabry mice and WT
controls (n = 7). (C) Correlation between Gusb mRNA levels and Gusb activities in 18-month-old male mouse kidneys. (D) Gusb activities in urine from 14- to 16month-old male Fabry mice and WT controls (n = 10–14). (E and F) mRNA levels of cardiac IGF-1 (E) and kidney Kap (F) in homozygous female Fabry mice and WT
controls (n = 6–8). (G) AR reporter gene assay in suspension cultures of renal proximal tubules using adenovirus containing MMTV-Luciferase with and without coexpression of exogenous AR by Ad5hAR (n = 4). (H) Corrected enzyme deficiency in IMFE1(α-gal+) cells. α-gal A protein detected by western blot (upper) and enzyme
activities (lower, unit: nmol/mg/h) are shown. (I) Gb3 immunostaining (red, Gb3; blue, DAPI nuclear counterstain). Gb3 accumulation was cleared in IMFE1(α-gal+) cells.
(J) PSA mRNA levels in IMFE1(mock) and IMFE1(α-gal+) cells (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 determined by t-test.
transcription, a Gb3-loading study was carried out in HL-1 cells,
an immortalized mouse cardiac cell line that retains a number
of characteristics of cardiomyocytes (30). HL-1 cells have normal
α-gal A activity and have no Gb3 detected by immunostaining
(Fig. 3E). It is known that exogenously added GSLs can be accumulated in plasma membrane and intracellular membrane systems
in cultured cells with normal lysosomal enzymes (31). Gb3 loading led to robust cell-associated ( presumably plasma membrane
and endosomes) accumulation of Gb3 in HL-1 cells (Fig. 3E), and
Gb3-loaded HL-1 cells had significantly increased AR mRNA expression compared with mock-treated cells (Fig. 3E). AR transcription was also upregulated in Fabry patients’ cells. AR
mRNA level was slightly but significantly increased in male
Fabry patients’ fibroblasts compared with healthy controls’ cells
(Fig. 3F) and was >14 times higher in IMFE1(mock) cells than in
IMFE1(α-gal+) (Fig. 3G).
Blockade of AR signaling ameliorates disease phenotypes
in male Fabry mice
We next examined whether increased activity of AR signaling is
responsible for disease pathogenesis. To this end, we tested
whether blockage of AR signaling by castration can prevent
the development of disease phenotype. Fabry male mice were
castrated at 2 months of age (an asymptomatic stage), and the
effects of castration were evaluated at 18 months of age.
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Figure 2. Increased activity of AR signaling in Fabry disease. (A) mRNA levels of IGF-1 and TGF-β1 in hearts, and Gusb, Kap and Acsm3 in kidneys in 18-month-old
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levels in male mouse heart (B) and kidney (C) tissues measured by real-time RT–PCR (n = 6–9). (D) AR mRNA levels in 12-month-old female mouse hearts (n = 6). (E) The role
of Gb3 on AR mRNA expression in HL-1 cells. Left: immunostaining against Gb3; the most Gb3-loaded HL-1 cells showed cell-associated positive signal (red, Gb3; blue, DAPI
nuclear counterstain). Right: AR mRNA levels in HL-1 cells cultured in the presence or absence of 20 μ of Gb3/albumin complex for 5 or 21 days (n = 3–6; statistical analysis,
Gb3-loaded versus mock-treated). (F) AR mRNA levels in skin fibroblasts obtained from male adult Fabry patients and healthy controls (n = 27 and 11, respectively). (G) AR
mRNA levels in IMFE1(mock) and IMFE1(α-gal+) cells (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 determined by t-test.
As expected, blood testosterone levels in castrated male Fabry
mice were markedly decreased and were even lower than in intact female Fabry mice (Fig. 4A). Compared with untreated male
Fabry mice, the mRNA levels of IGF-1 decreased and TGF-β1 increased in castrated Fabry mouse hearts (Fig. 4B). Expression of
Gusb, Kap and Acsm3 all decreased in castrated Fabry mouse
kidneys (Fig. 4C). Gusb activity in kidney decreased to the untreated WT level as well (Fig. 4D). Abolished expression of Kap
and Acsm3 in castrated mice indicated that AR signaling was
blocked by castration. Moreover, the restored expression of
IGF-1, TGF-β1 and Gusb by castration suggested that the aberrant expression of these genes in Fabry mouse hearts and kidneys (Fig. 2A) is indeed the consequence of enhanced AR
signaling. Castration completely prevented the development
of cardiac hypertrophy in Fabry mice. Heart-to-body weight
ratio, LV wall thickness measured by echocardiography and cardiac ANP mRNA levels in castrated Fabry mice were at the levels
of untreated WT mice (Fig. 4E–H). Akt phosphorylation in castrated
Fabry mouse hearts decreased significantly compared with untreated Fabry mice (Fig. 4I). Kidney weight of castrated Fabry
mice remained at WT levels as well (Fig. 4J). More importantly, in
WT mice, all these parameters were unchanged by castration
(Fig. 4E–J), suggesting that the effect of androgen depletion in
Fabry mice is disease process specific, rather than a universal
effect on normal heart and kidney development.
It is known that synthesis of Gb3 in murine kidneys is upregulated by androgens (32). To address whether the effect of castration on disease phenotypes of Fabry mice is through reduced
synthesis of Gb3, we measured Gb3 in mouse tissues. Gb3 was significantly reduced in castrated WT kidneys as expected (Fig. 4L)
but was unchanged in the hearts and kidneys of castrated
Fabry mice (Fig. 4K and L), indicating that the abolished cardiac
and kidney hypertrophy in castrated Fabry mice was not due to
reduced Gb3 in these organs.
Next, we tested whether castration can correct preexisting cardiac and renal pathology in Fabry disease. Fabry mice
were castrated at 12 months of age when cardiac and kidney
hypertrophy is apparent, and the effects were evaluated at 18
months of age. Heart weight, ANP mRNA level and kidney
weight in castrated Fabry mice were reduced to the levels
of untreated WT (Supplementary Material, Fig. S5A–C). Histologically, renal cortex width and proximal tubule wall thickness in castrated Fabry mice were reduced to untreated WT
levels (Supplementary Material, Fig. S5D and E). Importantly,
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Figure 3. Upregulated AR mRNA expression in Fabry disease. (A) AR in mouse heart homogenates (18-month-old males) was enriched by immunoprecipitation and was
detected by western blot. Whole lysate of mouse testis (25 μg total protein) was used as positive control. Data represent two independent experiments. (B and C) AR mRNA
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Figure 4. Castration abolishes disease phenotypes in male Fabry mice without changing the level of accumulating Gb3. (A) Blood testosterone levels in 3-month-old intact
or castrated male Fabry mice (4 weeks after castration) and untreated homozygous female Fabry mice (n = 6–8). (B) mRNA levels of IGF-1 and TGF-β1 in the hearts of
castrated and untreated Fabry mice (n = 6–9). (C) mRNA levels of Gusb, Kap and Acsm3 in the kidneys of castrated and untreated mice (n = 7). (D) Gusb activities in the
kidneys of castrated and untreated mice (n = 7–9). (E) Heart-to-body weight ratio (n = 9–12). (F) LV wall thickness measured by echocardiography (n = 3–7 per group). (G)
Representative M-mode echocardiography demonstrating abolished hypertrophy in castrated Fabry mice. Vertical bars indicate the thickness of anterior and posterior
walls. (H) ANP mRNA levels (n = 6–9). (I) Phosphorylation of Akt in castrated and untreated Fabry mouse heart tissues (n = 6–7). (J) Kidney-to-body weight ratio (n = 9–12). (K
and L) Gb3 levels in heart (K) and kidney (L) tissues (n = 5–6). *P < 0.05, **P < 0.01, ***P < 0.001 determined by t-test (A–D, I, K and L) or one-way ANOVA followed by Bonferroni’s
correction for multiple comparisons (E, F, H and J).
supplementation of exogenous androgen (DHT) in castrated
Fabry mice restored all these abnormalities (Supplementary
Material, Fig. S5A–E). This further confirmed the pathogenic
role of AR signaling in Fabry disease. The vacuolization of
proximal tubules, which may reflect glycolipid storage, was
not changed by castration.
Human Molecular Genetics, 2015, Vol. 24, No. 11
Discussion
As is the case in most lysosomal storage disorders, the downstream pathogenic mechanism of Fabry disease is complex.
Impaired membrane trafficking, autophagy, signaling transduction and inflammation process, and cellular injury due to toxic
metabolites (such as lyso-sphingolipids) are some of the common cellular abnormalities of sphingolipidoses (31,33–35). In
the present study, we proposed a new mechanism, i.e. that
enhanced AR signaling contributes to some of the clinical manifestations of Fabry disease.
The role of androgen action in cardiac hypertrophy is well established. Androgens induce a hypertrophic response in cultured
cardiomyocytes in an AR-dependent manner (16). Testosterone
administration in rats causes cardiac hypertrophy associated
with increased IGF-1 mRNA and Akt phosphorylation in the
heart with no significant change in ANP and β-MHC expression
and cardiac fibrosis (17). Conversely, AR-deficient mice show
smaller heart size and reduced hypertrophic response to angiotensin II stimulation (36). Angiotensin II causes more prominent
cardiac fibrosis and systolic dysfunction in AR-deficient mice
than in WT mice with increased TGF-β1 mRNA and Smad2 activation (36). These studies suggest that the androgen/AR system induces or promotes hypertrophic response probably through the
IGF-1/Akt signaling pathway but, on the other hand, suppresses
the development of fibrosis and systolic dysfunction via decreased activity of TGF-β1/Smad signaling. The pattern of androgen/AR-induced cardiac hypertrophy (i.e. less significant fetal
gene upregulation, cardiac impairment and fibrosis) clearly differs from that seen in experimental models of pressure overload-induced cardiac hypertrophy (37). Cardiac phenotypes of
Fabry mice are consistent with the pattern of androgen-induced
hypertrophy. The generally preserved ejection fraction in Fabry
patients (4) is also in agreement with this pattern. A difference
between mice and humans is cardiac fibrosis, which is often
seen in Fabry patients. Possible reasons for this discrepancy are
the longer disease course in patients (years or decades) and/or
Figure 5. AR-antagonist ameliorates disease phenotypes in male Fabry mice. (A) Gusb enzyme activity in kidneys from flutamide- or vehicle-injected mice (n = 6–8). (B)
Heart-to-body weight ratio (n = 6–10). (C) LV mass analyzed by echocardiography (n = 5–8). (D) Kidney-to-body weight ratio (n = 6–10). *P < 0.05 determined by t-test.
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We further tested whether pharmacological blockade of AR
signaling can ameliorate disease phenotypes. Eighteen-monthold Fabry mice were treated with flutamide for 7 weeks by daily
subcutaneous injection at a dose of 25 mg/kg/day. Flutamide is
a potent non-steroidal AR-antagonist; it competes with testosterone and DHT for binding to AR. Gusb activity in kidney decreased
in both flutamide-injected WT and Fabry mice, with more significant decrement in Fabry mice (Fig. 5A) suggesting attenuated activity of AR signaling. Flutamide treatment resulted in decreased
heart-to-body weight ratio and LV mass as well as decreased kidney-to-body weight ratio in Fabry mice compared with placebotreated Fabry mice (Fig. 5B–D). Consistent with the findings in
the castration study (Fig. 4), these parameters in WT mice were
not changed by flutamide treatment (Fig. 5B and D).
Taken together, these results suggested that AR signaling
contributes to cardiac and renal hypertrophy in male Fabry
mice and that blockade of AR signaling is able to prevent and correct these disease phenotypes.
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The mechanism through which Gb3 accumulation causes increased activity of AR remains unclear. Transcriptional activity of
AR is subjected to complex regulation. It is regulated by a number
of coactivators and corepressors that influence functional properties of AR, including ligand selectivity and DNA binding capacity
(23). The increased transactivation activity of exogenous AR in the
reporter gene assay suggests abnormal AR coactivators in Fabry
mouse cells. AR activity is modulated by phosphorylation of AR or
its coactivators. Several signal transduction pathways such as
MAPK, Akt and PKC are known to be involved in this phosphorylation (43,44). On the other hand, our data showed increased AR
mRNA in Fabry disease. AR promoter activity is regulated by several
transcription factors (45–48), whose activities are modulated by different signaling pathways such as Akt and Wnt. We hypothesize
that both increased AR mRNA and activated AR signaling might
be mediated by the same upstream signaling pathway. Close correlation between mRNA levels of AR and androgen-regulated genes
(Supplementary Material, Fig. S4) supports this possibility. If so, analysis of AR promoter activity in cultured cells might facilitate identification of the signaling pathway responsible for the AR activation.
The blocking of AR signaling was able to ameliorate disease phenotypes in male Fabry mice despite the persistence of α-gal A
deficiency and GSL storage suggesting that AR signaling is an important downstream pathway to GSL storage and can be a useful
molecular target for therapy. The reversal of cardiac and renal
hypertrophy in castrated or flutamide-treated Fabry mice suggested
that AR signaling plays roles not only in development but also
maintenance of these disease phenotypes. Although we used flutamide in animal studies, because of the agonist properties of flutamide (49), the second-generation antiandrogens such as MDV3100
(50) would be more suitable for potential therapy in humans. AR
pathway-targeted approaches have been extensively studied for
treating prostate cancer (51); thus, there are a number of agents
available for clinical use. These strategies may be used as new therapy for Fabry disease or can be used in combination with preexisting therapies (e.g. ERT) to obtain better therapeutic efficacy.
Materials and Methods
Mice and treatments
All mice used in this study were maintained in our laboratory
under standard housing conditions. Fabry mice (14) were produced by breeding pairs of hemizygous males and homozygous
females. To determine genetic background, we performed microsatellite analysis (by Charles River), and the results showed that
Fabry mice are mixtures of C57BL/6J and 129 strains with ∼75% of
C57BL/6J strain background. WT mice with the same strain background were used as controls throughout the studies. All procedures performed on the mice were reviewed and approved by
the Institutional Animal Care and Use Committee of Baylor Research Institute. Castration was performed under anesthesia
using ketamine and xylazine. For androgen replacement, timerelease DHT pellets (25 mg/pellet for 90 days release, Innovative
Research of America) were implanted subcutaneously 1 week
after castration. The pellets were replaced every 3 months. For
flutamide treatment, flutamide dissolved in 10% ethanol in
corn oil was injected subcutaneously daily at a dose of 25 mg/
kg/day. Control mice received vehicle injection.
Echocardiography
In vivo cardiac morphology was assessed by transthoracic
echocardiography (Vevo 2100, VisualSonics) in conscious mice.
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differences in signaling systems that are modulated by AR in the
mouse and human heart.
It has long been recognized that the kidney is an androgensensitive organ (18,19). Androgens cause hypertrophy and increased expression of Gusb and other specific proteins in murine
kidneys (18,19,26). Proximal tubule cells are the predominant cell
type responsible for androgen action in kidneys with respect to
renal hypertrophy and induction of androgen-regulated genes
(19). Our results revealed that the cellular basis of kidney enlargement in Fabry mice is hypertrophied proximal tubule cells
mediated by AR signaling. Enlarged kidneys in Fabry patients (5)
are probably caused by the same mechanism. Activated proximal
tubules can be involved in kidney disease pathogenesis via various mechanisms including generation of reactive oxygen species
and inflammatory cytokines (38). However, because Fabry mice do
not develop renal dysfunction, it is difficult to investigate whether
and how the proximal tubule hypertrophy contributes to Fabry
kidney disease. A recent study reported that Fabry mice with increased Gb3 synthesis develop renal impairment associated
with significantly increased kidney-to-body weight ratio in both
sexes (39). It will be important to study the effects of AR-targeting
treatment in renal dysfunction in this Fabry mouse strain.
Although our interventional studies were focused on male
Fabry mice, we hypothesize that AR contributes to Fabry disease
in females as well. First, although androgens are not dominant
sex hormones in females, AR activity in hearts and kidneys of female mice is similar to that in males (40). Thus, aberrant AR signaling can be pathogenic in females. Second, homozygous female
Fabry mice develop similar phenotypes to those found in male
Fabry mice, suggesting that both sexes share the same pathogenic
mechanism. Third, the upregulated IGF-1 suggested increased AR
activity in female Fabry mouse hearts. However, given the different hormone systems, it is possible that some different mechanisms may be operational in females compared with males. This
was suggested by the sexual dimorphism in the gene expression
profile of Fabry mouse kidneys, typically in Kap expression. Kap
is strictly regulated in a sex-specific manner (41). In males, it is
expressed in the S1/S2 segments of proximal tubules and is regulated by androgens. In contrast, in females it is exclusively expressed in the S3 segment and is regulated not only by androgens,
but also by thyroid hormone and estrogen. Thus, expression of Kap
in female kidneys is the result of combined effects of multiple hormones and may not be a good indicator of AR activity.
It should be noted that the conclusion obtained from homozygous female Fabry mice cannot be directly applied to the pathogenesis of cardiac hypertrophy in heterozygous female patients,
in which the heart is a mosaic of normal and affected cells resulting from random X-chromosome inactivation. It is intriguing
as to how dysfunction of the entire organ occurs when only
some of the cells are affected. One potential mechanism can be
paracrine action of IGF-1, i.e. the increased secretion of IGF-1
(and/or other growth factors) from affected cardiomyocytes
may act on adjacent normal cardiomyocytes, causing their
hypertrophic growth. Further investigations should be conducted
in heterozygous female Fabry mice to address this possibility.
Upregulated PSA in Fabry patients’ endothelial cells suggested that increased AR activity may be also present in human
patients. However, we cannot rule out the possibility that there
may be some species-specific disease mechanisms that are related to a different glycolipid metabolism in mice and humans,
e.g. the less profound GSL storage in Fabry mouse endothelial
and cardiac cells than in patients (42). Further investigations
will be necessary to gain more direct evidence for enhanced AR
signaling in Fabry patients.
Human Molecular Genetics, 2015, Vol. 24, No. 11
Systolic and diastolic LV internal dimensions and LV wall thickness were averaged from 3–6 beats. Thicknesses of anterior and
posterior walls were averaged. The analysis was performed in a
manner blind to genotype and treatment conditions.
Kidney histology
Kidneys were cut transversely, fixed in 10% formaldehyde and
paraffin embedded. Five-micrometer-thick sections were made
and stained with PAS. Photomicrographs were analyzed using
imaging software to assess cortical width and proximal tubule
wall thickness. The analysis was performed by a veterinarian
pathologist (M.W.C.) in a blinded manner.
Urine analysis
Cell culture and treatments
Skin fibroblasts obtained from male Fabry patients (n = 27, mean
donor age 39.7 with average residual α-gal A activity in leukocytes
3.6% of normal) and male normal controls (n = 11, mean age 27.3)
were cultured in 10% fetal bovine serum (FBS) in DMEM.
Mouse cardiac muscle cell line, HL-1 (30), was cultured in
Claycomb basal medium (Sigma) supplemented with 10% FBS,
0.1 m norepinephrine and 2 m -glutamine. Gb3 loading was
performed as previously described (28). The cells were incubated
with 20 μ Gb3/albumin complex for 5 or 21 days before analysis.
Gb3 accumulation in the cells was confirmed by Gb3 immunostaining. For mock treatment, solvent (DMSO) was used in place
of Gb3.
IMFE1 cells, a Fabry patient-derived microvascular endothelial cell line (28), were cultured in EGM2MV medium (Lonza). To
correct the enzyme deficiency, IMFE1 cells were infected with a
MMP retroviral vector encoding normal α-gal A with co-expression of GFP through an internal ribosome entry site (IRES) (52).
Control cells [IMFE1(mock)] were infected with an empty retroviral vector (the same backbone without α-gal A gene). The infected cells were purified by selecting GFP-positive cells using
fluorescence-activated cell sorting. After sorting, the GFP-positive cell purity was ∼93% for both IMFE1(α-gal+) and IMFE1(mock).
Culture of proximal tubules and AR reporter gene assay
Renal proximal tubules were isolated from 12-month-old male
Fabry and WT mice as described previously (53) with modifications. Briefly, renal cortices were dissected and minced into
small pieces (∼1 mm3) and were digested with 1 mg/ml type-2
collagenase (Invitrogen) at 37°C for 30 min in the presence of
1 mg/ml soybean trypsin inhibitor (Sigma). The same volume of
horse serum was added, and the suspension was mixed by vortex
for 30 s. The suspension was allowed to sediment for 1 min, and
the proximal tubule-enriched supernatant was centrifuged at
200g for 7 min. After being washed with HBSS to remove serum,
the tubule fragments were suspended in DMEM/F12 (1:1) supplemented with 5% charcoal-stripped FBS (cs-FBS) and were plated
on 48-well plates. Recombinant adenoviruses Ad/MLUC7 (reporter vector containing MMTV-Luciferase (27); 1 × 106 pfu) and Ad-
LacZ (internal control for gene transduction efficiency; 0.25 × 106
pfu) were added to each well. Ad5hAR (27) was also added to
some wells (0.25 × 106 pfu/well) to express exogenous AR. The infection was performed in the volume of 0.1 ml/well for 3 h with
occasional agitation. After that, 5% cs-FBS in DMEM/F12 and
DHT were added to achieve the final volume of 0.2 ml/well with
or without 10 n DHT. Two days later, the tubules were harvested
and lysed for reporter assays. Luciferase activity was determined
using Luciferase reporter assay kit (Promega) and a luminometer
(GloMax 20/20, Promega). β-Galactosidase activity was measured
using the colorimetric method with 1 mg/ml o-nitrophenyl-β-galactopyranoside at pH 7.0. Luciferase activity was normalized
by β-galactosidase activity.
Quantitative RT–PCR
Quantitative RT–PCR was performed as described previously (28),
using pre-designed TaqMan probe/primers (Applied Biosystems)
or SYBR Green primers (Sigma). 18S rRNA was used as internal
control.
PCR array
The expression profile of 84 genes related to endothelial biology
was analyzed by using Human Endothelial Cell Biology PCR
Array kit (PAHS-015A-24, SABiosciences). Four independent assays were performed. For each assay, a pair of IMFE1(α-gal+)
and IMFE1(mock) cells were cultured in parallel and were subjected to RNA extraction and reverse transcription. Quantitative
PCR was performed using StepOne Real-time PCR system (Applied Biosystems) and the fold-change was calculated using
PCR Array analysis software (SABiosciences).
Western blot and immunoprecipitation
Western blot analysis was performed as described previously
(28). Primary antibodies used were rabbit antibodies to phospho-Akt (Ser473), total Akt (Cell Signaling), AR (N-20, Santa
Cruz) and human α-gal A (Shire), and goat antibody to GAPDH
(Santa Cruz). Intensity of the bands was analyzed using ImageJ
software. Because of the low abundance, AR protein in mouse
heart and kidney lysates could not be directly detected by western blot, and thus, AR was enriched by immunoprecipitation. In
brief, mouse tissues were homogenized in RIPA buffer with protease inhibitors (Santa Cruz). Lysates containing 1 mg total protein were incubated with 1 μg rabbit antibody to AR (N-20, Santa
Cruz) overnight at 4°C followed by mixing with 20 μl Protein A/G
PLUS-Agarose beads (Santa Cruz) for 1 h at 4°C. For negative control, normal rabbit IgG was used. The beads were washed four
times with 0.5% Triton-X in PBS, and the proteins on the beads
were analyzed by western blot using the antibody to AR.
Testosterone level
Plasma level of total or free testosterone was measured using
radioimmunoassay kit (Siemens Diagnostics).
Analysis of Gb3 levels
Gb3 levels in mouse tissues were measured by mass spectrometry as described previously (54). Gb3 levels in cultured cells
were analyzed by immunostaining using monoclonal antibody
to Gb3 as described (28).
Downloaded from http://hmg.oxfordjournals.org/ at Pennsylvania State University on September 18, 2016
Mouse overnight urine samples were collected using metabolic
cages and were stored at −80°C until use. Urine albumin concentration was measured using a mouse albumin ELISA kit (Bethyl
Lab). For Gusb activity, 10 μl of urine was directly used for enzyme
assay. Urinary albumin concentrations and Gusb enzyme activities were normalized by creatinine concentrations that were
measured using COBAS Integra 400.
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Lysosomal enzyme assays
Activities of α-gal A and Gusb were determined by the fluorimetric method using 4-methylumbellferyl-labeled α--galactopyranoside and β--glucuronide, respectively.
10.
Statistical analysis
Data were presented as mean ± s.e.m. Statistical significance was
determined by the Student’s t-test. One-way ANOVA followed by
Bonferroni’s test was used for multiple comparisons.
11.
Supplementary Material
12.
Supplementary material is available at HMG online.
We thank P.C. Hartig (US Environmental Protection Agency, Research Triangle Park, NC) for providing the adenovirus vectors,
Ad/mLuc7 and Ad5hAR; W.C. Claycomb (Louisiana State University Medical Center, New Orleans, LA) for providing HL-1 cell line;
H. Tabata (Keio University School of Medicine, Tokyo, Japan) for
help in cell sorting; and C. Chang (University of Rochester Medical
Center, Rochester, NY), L. Sweetman and T. Bottiglieri (Baylor
Research Institute, Dallas, TX) for critical discussions and
suggestions.
13.
14.
15.
Conflict of Interest statement. None declared.
Funding
16.
This work was supported by the Baylor Health Care System
Foundation.
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