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Am J Physiol Cell Physiol 293: C805-C813, 2007. First published May 16, 2007; doi:10.1152/ajpcell.00127.2007
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VASCULAR BIOLOGY

PKC{delta} mediates anti-proliferative, pro-apoptic effects of testosterone on coronary smooth muscle

D. K. Bowles,1,2,3 K. K. Maddali,1,5 V. C. Dhulipala,6 and D. H. Korzick4

1Department of Biomedical Sciences, 2Dalton Cardiovascular Research Center, 3National Center for Gender Physiology, University of Missouri, Columbia, Missouri; 4Intercollege Program in Physiology, The Pennsylvania State University, University Park; and 5Resources and 6Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania

Submitted 29 March 2007 ; accepted in final form 15 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sex hormone status has emerged as an important modulator of coronary physiology and cardiovascular disease risk in both males and females. Our previous studies have demonstrated that testosterone increases protein kinase C (PKC) {delta} expression and activity in coronary smooth muscle (CSMC). Because PKC{delta} has been implicated in regulation of proliferation and apoptosis in other cell types, we sought to determine if testosterone modulates CSMC proliferation and/or apoptosis through PKC{delta}. Porcine CSMC cultures (passages 2–6) from castrated males were treated with testosterone for 24 h. Testosterone (20 and 100 nM) decreased [3H]thymidine incorporation in proliferating CSMC to 59 ± 5.3 and 33.1 ± 4.5% of control. Flow cytometric analysis demonstrated that testosterone induced G1 arrest in CSMC with a concomitant reduction in the S phase cells. Testosterone reduced protein levels of cyclins D1 and E and phosphorylation of retinoblastoma protein while elevating levels of p21cip1 and p27kip1. There were no significant differences in the levels of cyclins D3, CDK2, CDK4, or CDK6. Testosterone significantly reduced kinase activity of CDK2 and -6, but not CDK4, -7, or -1. PKC{delta} small interfering RNA (siRNA) prevented testosterone-mediated G1 arrest, p21cip1 upregulation, and cyclin D1 and E downregulation. Furthermore, testosterone increased CSMC apoptosis in a dose-dependent manner, which was blocked by either PKC{delta} siRNA or caspase 3 inhibition. These findings demonstrate that the anti-proliferative, pro-apoptotic effects of testosterone on CSMCs are substantially mediated by PKC{delta}.

androgens; coronary; smooth muscle; cell cycle

MEN 30–50 YR OF AGE have an increased incidence of coronary artery disease (CAD) compared with women of similar age (2, 4, 30). This sex difference in the prevalence of CAD led to the widespread belief that testosterone increases the risk of heart disease in men. However, recent clinical studies have failed to support a detrimental effect of testosterone on the incidence or severity of CAD (17, 46) or carotid atherosclerosis (24) in men. On the contrary, a growing body of epidemiological and clinical trial data indicates that low testosterone levels in men are associated with a higher risk of cardiovascular disease (55). For example, both low testosterone levels and free androgen index have been reported in men with CAD (17, 20, 44, 46), aortic atherosclerosis (26), and carotid atherosclerosis (41). Similarly, low testosterone levels are associated with increased risk factors for cardiovascular disease, specifically obesity, hypertension, hyperglycemia, and hypercholesterolemia (24, 41, 46), suggesting that testosterone may limit the progression of CAD indirectly through beneficial modification of risk factors, independent of direct androgenic actions on the vascular wall. However, in an in vitro organ culture model, testosterone produced significant reductions of neointimal plaque development in aortas of male rabbits (28), supporting a direct effect of testosterone on the vascular cells. Additionally, local conversion of testosterone to estrogen via aromatase has been proposed to mediate potentially beneficial effects of testosterone (3, 57).

Accumulation of smooth muscle cells (SMC) in the intima is a hallmark of coronary atherosclerosis and postangioplasty restenosis (47). Because SMC proliferation and apoptosis coincide in arteriosclerotic lesions, the balance between these two processes determine SMC accumulation during vascular remodeling and lesion development (5, 7, 38). SMC proliferation is tightly regulated by the complex interaction of numerous cell cycle regulatory proteins at specific check points of cell growth (47). These cell cycle regulatory proteins are influenced by kinase signaling pathways, including protein kinase C (PKC; see Ref. 25). Overexpression of PKC{delta} inhibits growth rates and proliferation of rat aortic SMCs (25, 34). In vivo, PKC{delta} null mice demonstrate exacerbated vein graft arteriosclerosis indicating a critical role for PKC{delta} in regulating vascular SMC differentiation and proliferation (34). The ability of PKC{delta} to suppress the expression of positive regulatory factors required for the cell cycle progression indicates that PKC{delta} has gatekeeper functions similar to those of other known tumor suppressor genes, such as retinoblastoma protein (Rb; see Ref. 25). In addition, recent studies show that cells derived from PKC{delta} null transgenic mice are defective in mitochondrial-dependent apoptosis (34). Proteolytic activation of PKC{delta} by caspases, which results in the generation of an active kinase domain, occurs in response to a variety of pro-apoptotic stimuli, including DNA-damaging agents (19, 45), FAS ligand (22), and mitomycin C (18). Interestingly, when the catalytic domain of PKC{delta} is transiently transfected into cultured cells, it rapidly induces apoptosis (6, 39). Therefore, factors that alter levels of PKC{delta} in vascular smooth muscle have the potential to affect the accumulation of smooth muscle in vasculoproliferative diseases by altering the balance between proliferation and apoptosis.

We have recently shown that 1) PKC{delta} levels are higher in coronary smooth muscle of males compared with females, 2) endogenous testosterone increases PKC{delta} protein levels in coronary smooth muscle of swine, and 3) both testosterone and dihydrotestosterone (DHT) increase PKC{delta} expression and activity in coronary smooth muscle cells (CSMC) in vitro (33, 35). Given the evidence for an important regulatory role of PKC{delta} in cell proliferation and apoptosis of noncoronary cell types, the purpose of this study was to determine if testosterone-mediated increases in PKC{delta} affect CSMC proliferation and/or apoptosis. Our results demonstrate that testosterone induced a PKC{delta}-dependent G1/S phase cell cycle arrest and stimulated apoptosis, providing a potential mechanistic basis for observed effects of testosterone on coronary vasculoproliferative diseases, such as postangioplasty restenosis and atherosclerosis (49).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Porcine CSMC culture. Cultures of porcine medial CSMC were performed as previously described (35). After removal of the adventitia and endothelium, CSMCs were isolated from the remaining media by enzymatic dispersion and plated at 1.5 x 104 cells/cm2 in DMEM/F-12 media (GIBCO 11320-033), 100 U/ml penicillin/streptomycin, 1.6 mM L-glutamine, and 10% FBS for 2–3 days, changing media every 2 days. Upon attaining 40–50% confluence, the CSMC were treated with testosterone (20 or 100 nM) or vehicle (100% ethanol) in phenol red-free DMEM medium with 10% charcoal-stripped serum at 37°C for a period of 24 h. Experiments were performed on cells from passages 2–6 as previously we have noted increased variability in responses to hormones beyond passage 6. Cell growth was determined by hemocytometer. Gene silencing of PKC{delta} using small interfering RNA (siRNA) was performed as previously described (35).

[3H]thymidine incorporation. CSMC were pulse labeled with [3H]thymidine (2 µCi/ml) for 4 h before a 24-h testosterone or vehicle exposure period. Average [3H]thymidine incorporation was expressed as counts per well.

Cell cycle analysis. Flow cytometric analysis was performed as previously described (25). Upon attaining 40–50% confluence, CSMC were treated with testosterone (20 and 100 nM) for 24 h, subjected to propidium iodide (PI) staining, and analyzed by fluorescence-activated cell sorter (FACS) analysis.

Cyclin-dependent kinase activity assays. Kinase activities were performed for cyclin-dependent kinases (CDKs) as previously described (16).

Immunoblot. Immunoblots were performed as previously described (8, 16, 35). CSMC lysates were obtained in lysis buffer containing 1% Triton X-100. Equal amounts of sample per lane (30–60 µg protein) were subjected to electrophoresis and transferred to a polyvinylidene difluoride membrane. beta-Actin was used as a loading control. All antibodies were obtained from Santa Cruz Biotechnologies except CDK2 and CDK7 (Upstate).

Caspase 3 activity assay. The Enzchek caspase 3 assay kit by Molecular probes (Eugene, OR) was used according to the manufacturer's instructions to determine the amount of apoptosis in CSMC cultures. Cells were collected and analyzed by Enzchek caspase 3 assay kit no. 2, using a HITACHI F4010 fluorescence spectrophotometer (496/520 nm). This kit provides the substrate of caspase 3 Z-DEVD-R110, which can be lysed by active caspase 3 and release R110, the fluorescence of which was analyzed by fluorescence spectrophotometer. Briefly, 2 x 106 PKC{delta} siRNA-treated cells, with and with out testosterone treatment, were collected after 24 h incubation in 10% FCS and washed in PBS, and then the lysates were assayed for caspase 3 activity. Exposure to ceramide (2.5 µM) was used as a positive control for apoptosis.

Annexin V analysis. Annexin V analysis was performed as described previously (21). Upon attaining 40–50% confluence, CSMC were treated with testosterone (20 and 100 nM) for 24 h and washed and incubated with FITC-labeled annexin V in binding buffer (Roche) for 20 min in the dark. Cells were washed and resuspended in 200 µl PBS and PI (1 mg/ml), and a nuclear dye solution was added. Flow cytometry was performed in a FACScan. Annexin V staining was also analyzed in PKC{delta} siRNA cells, treated with and with out testosterone using flow cytometric analysis.

TdT-UDP nick end labeling assay. DNA strand breaks typical for apoptosis were detected using the Fluorescein In Situ Cell Death Detection Kit (Roche). Cells were trypsinized, fixed (2% paraformalydhyde), and permeabilized for 30 min using 70% ethanol on ice. TdT-UDP nick end labeling (TUNEL) staining was examined by confocal microscopy. The percentage of positively stained cells was determined by counting the numbers of labeled and total cells.

Statistical analysis. All data are presented as means ± SE. Statistical significance among treatment groups was determined using ANOVA with Bonferroni post hoc analyses. A P value ≤0.05 was set as the criterion for significance in all comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Testosterone inhibits CSMC proliferation via PKC{delta}. CSMC numbers were reduced in a concentration-dependent manner following 24 h of testosterone treatment (Fig. 1A). Compared with vehicle-treated controls, CSMC numbers were decreased by 25 and 40% by 20 and 100 nM testosterone, respectively. Similar effects of testosterone were observed on [3H]thymidine incorporation whereby 20 and 100 nM testosterone suppressed [3H]thymidine incorporation to 59 ± 5.3 and 33.1 ± 4.5% of control (Fig. 1B). DHT produced a reduction in [3H]thymidine incorporation similar to testosterone (Fig. 1D). In vivo, testosterone can be converted to either estrogen or DHT by aromatase or 5-{alpha} reductase, respectively. DHT is a nonaromatizable androgen that acts through binding to the androgen receptor. The similar effects of testosterone and DHT on [3H]thymidine incorporation provide strong evidence that testosterone conversion to estrogen by aromatization is not necessary for these observed effects of testosterone on CSMC proliferation.


Figure 1
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  		Fig. 1. Testosterone inhibits coronary smooth muscle cell (CSMC) proliferation. CSMC (3,000/cm2) were plated in phenol red-free DMEM medium containing 10% charcoal-stripped FBS and grown until 40–50% confluence (72 h). Testosterone (T; 20 and 100 nM) was added in the medium for an additional 24 h. Testosterone produced a concentration-dependent decrease in CSMC proliferation as indicated by decreased cell numbers/well (A) and [3H]thymidine incorporation/well (B). P < 0.05 vs. 0 nM (*) and vs. 0 and 20 nM (#). To evaluate the role of protein kinase C (PKC) {delta}, small interfering RNA (siRNA) was used against PKC{delta} as previously described and validated (35). CSMC were transfected either with control (scrambled) siRNA (filled bars) or PKC{delta} siRNA (gray bars) before treatment with testosterone. PKC{delta} knockdown almost completely blocked the inhibition of proliferation as measured by both cell number (C) and [3H]thymidine incorporation (D). Dihydrotestosterone (DHT) and testosterone (100 nM) demonstrated similar PKC{delta}-dependent effects on proliferation (D). Inset: immunoblot of CSMC treated with control (–{delta}) and PKC{delta} (+{delta}) siRNA probed for PKC{delta} protein. beta-Actin was probed as a loading control. Autoradiograms are representative of 4 independent experiments. Value nos. are expressed as means ± SE; n = 4 experiments/treatment. *P ≤ 0.05 vs. respective control (C). {dagger}P ≤ 0.05, PKC{delta} siRNA vs. control siRNA.

 
We have previously validated the efficacy and specificity of PKC{delta} siRNA in our CSMCs, whereby PKC{delta} siRNA lowered PKC{delta} protein levels without altering the expression of PKC{alpha}, -{varepsilon}, or -{zeta} (35). Similarly, in this study, successful RNA silencing was demonstrated by ~90% knock down of PKC{delta} protein (Fig. 1D, inset). Silencing PKC{delta} greatly inhibited, but did not completely reverse, the effect of testosterone on cell number (Fig. 1C). PKC{delta} siRNA reversed both the testosterone and DHT-induced inhibition of thymidine incorporation (Fig. 1D). However, testosterone still produced a small inhibition proliferation after PKC{delta} knockdown with siRNA, as determined from both cell number and [3H]thymidine incorporation, suggesting a minor PKC{delta}-independent mechanism of testosterone on proliferation. Together, these data support the hypothesis that PKC{delta} provides a major pathway for testosterone-induced inhibition of CSMC proliferation.

Testosterone induces CSMC G1/S arrest via a PKC{delta}-dependent mechanism. To further elucidate the mechanism of testosterone on cell cycle progression, we performed flow cytometry on CSMC with and without PKC{delta} siRNA (Fig. 2). Testosterone produced a concentration-dependent increase in G0/G1 phase cells and decreased the number of S phase cells (Fig. 2). Conversely, testosterone had no effect on the distribution of cells in G0/G1, S, or G2/M in CSMCs treated with PKC{delta} siRNA. Thus testosterone suppresses cell proliferation by inhibiting the G1-to-S transition in a PKC{delta}-dependent manner, supporting the hypothesis that PKC{delta} acts as a gatekeeper mediating testosterone effects on CSMC proliferation.


Figure 2
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  		Fig. 2. Testosterone induces CSMC G1/S arrest in a PKC{delta}-dependent mechanism. CSMC (3,000/cm2) were plated in phenol red-free DMEM medium containing 10% charcoal-stripped FBS and grown until 40–50% confluence (72 h). Testosterone (20 and 100 nM) was added in the medium for an additional 24 h. Cells were fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry using fluorescence-activated cell sorter analysis scan (FACScan). Parallel experiments were performed with CSMC treated with control and PKC{delta} siRNA. Testosterone caused a G1/S block as demonstrated by a reduction in the number of S phase cells with a corresponding increase in the G0/G1 phase cells compared with the control group. PKC{delta} siRNA reversed the testosterone-induced G1/S arrest in a concentration-dependent manner. Values are means ± SE (n = 4). *P < 0.05 vs. 0 nM. {dagger}P ≤ 0.05 vs. 0 and 20 nM.

 
Regulation of G1/S cell cycle proteins by testosterone. We next examined the potential mechanisms underlying testosterone suppression of cell proliferation via PKC{delta}. The observation that testosterone produces G1/G0 phase arrest via PKC{delta} led us to examine the effect of testosterone on expression of specific G1 cyclins and CDKs necessary for transition into S phase (Fig. 3). We also examined the effects of testosterone on the CDK inhibitors (CDKI) p21cip1 and p27kip1. Testosterone produced a concentration-dependent reduction in protein levels of the positive cell cycle regulators cyclin D1 and E (Fig. 3, A and B), with no effects on the expression of cyclin D3 protein levels (Fig. 3C). Testosterone also produced a concentration-dependent increase in the protein levels of p21cip1, whereas increases in p27kip1 were similar at both concentrations (Fig. 3, D and E). Cyclin D- and E-associated CDKs act, in part, to increase levels of phosphorylated retinoblastoma (pRb), a necessary final step for cell cycle progression (10, 32, 34, 43). In accordance with the downregulation of cyclin D1 and cyclin E, pRb phosphorylation was reduced in a concentration-dependent manner by testosterone (Fig. 3F). These data demonstrate that the testosterone-induced reduction in CSMC proliferation is associated with a delayed G1/G0 exit resulting from decreased cyclin D1, E, and pRb and increased p21cip1 and p27kip1 protein levels.


Figure 3
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  		Fig. 3. Testosterone regulation of various CSMC G1/S cell cycle proteins. Densitometric analysis for effects of testosterone (20 and 100 nM) on protein levels of cyclin D1 (A), cyclin E (B), cyclin D3 (C), p21cip1 (D), p27kip1 (E), and phosphorylated retinoblastoma (pRb) protein (F). Insets: representative immunoblots from corresponding experiment. Testosterone produced a concentration-dependent decrease in cyclin D1 and E and pRb and an increase in p21cip1 and p27kip1, although both concentrations of testosterone were equally effective at increasing the latter. Values are means ± SE; n = 4–5. P < 0.05 vs. 0 nM (*) and vs. 0 and 20 nM ({dagger}).

 
Testosterone inhibits kinase activity of G1/S CDKs in CSMC. Testosterone had no effect on protein levels of CDK proteins 2, 4, and 6 (Fig. 4, A–C). Because CDK2, -4, and -6 are activated in proliferating cells, measuring the kinase activities of these proteins is critical for determining the effects of testosterone on CSMC proliferation. Immunoprecipitates of CDK1, -2, -4, -6, and -7 from CSMC treated with 20 and 100 nM testosterone were incubated with CDK-specific substrates (synthetic Rb peptide for CDK4/6 and histone H1 for CDK1, -2, and -7), and the phosphorylated levels of substrates were determined. Testosterone inhibited CDK2 and -6 activities in a concentration-dependent manner (Fig. 4, D and F), whereas CDK4 activity was unchanged (Fig. 4E). Testosterone had no effect on the activity of CDK7 or CDK1, a G2/M phase-associated kinase (data not shown). These data demonstrate that testosterone specifically inhibits the activity of G1/S transition of CDK2 and CDK6 in CSMC.


Figure 4
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  		Fig. 4. Testosterone decreases coronary cyclin-dependent kinase (CDK) 2 and CDK6 activity in CSMC. Protein and kinase activity assays were performed for CDK2, CDK4, and CDK6. Total protein for each CDK was unchanged by testosterone (A–C). However, gel kinase activity assays demonstrated a concentration-dependent decrease in kinase activity for CDK2 (D) and CDK6 (F). Testosterone had no effect on CDK4 (B) nor CDK1 or -7 kinase activities (data not shown). Insets: representative autoradiograms from corresponding experiments. CSMC were incubated for 24 h in the absence or presence of testosterone. Values are means ± SE. P < 0.05 vs. 0 nM (*) and vs. 0 and 20 nM ({dagger}) testosterone (n = 4–5).

 
Testosterone, CSMC cell cycle proteins, and PKC{delta}. As shown in Fig. 2, PKC{delta} siRNA prevented the testosterone-induced G1/G0 arrest in CSMC. Thus we examined the role of PKC{delta} in testosterone downregulation of cyclin D1 and E and upregulation of p21cip1 and p27kip1 protein levels. Immunoblots were obtained from testosterone-treated CSMC with and without PKC{delta} knockdown. PKC{delta} siRNA completely inhibited the downregulation of cyclin D1 and partially inhibited the downregulation of cyclin E protein (Fig. 5). Furthermore, testosterone failed to increase the protein levels of p21cip1 in CSMC in the presence of PKC{delta} siRNA. On the contrary, PKC{delta} siRNA had no effect on the testosterone-induced increase in CSMC p27kip1 protein levels. Thus PKC{delta} is required for testosterone suppression of cyclin D1 and E and upregulation of p21cip1 but not p27kip1.


Figure 5
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  		Fig. 5. Testosterone decreases coronary cyclin D1 and E and increases p21cip1 protein levels through PKC-{delta}. Group densitometric values for the effect of PKC-{delta} siRNA on testosterone downregulation of cyclin D1 (A) and cyclin E (B) and upregulation of p21cip1 (C) and p27kip1 (D) protein (n = 6/condition). Insets: representative immunoblots for each corresponding experiment. PKC{delta} siRNA (gray bars) and control siRNA-treated (black bars) CSMC were incubated for 24 h in the absence or presence of 100 nM testosterone. PKC{delta} siRNA inhibited testosterone downregulation of cyclin D1 and E and upregulation of p21cip1 protein levels. PKC-{delta} siRNA failed to reverse testosterone-induced upregulation of p27kip1 protein levels in CSMC. Values are means ± SE. *P < 0.05 0 vs. 100 nM testosterone. {dagger}P ≤ 0.05, control vs. PKC{delta} siRNA.

 
Testosterone increases apoptosis via PKC{delta}. These data clearly show that testosterone inhibits CSMC proliferation through a PKC-{delta}-dependent mechanism. Next we sought to determine if testosterone increases CSMC apoptosis using annexin V assay. Annexin V binds to phosphatidylserine exposed on the surface of apoptotic cells while PI uptake identifies necrotic cells. Treatment of subconfluent CSMC with testosterone for 24 h a induced concentration-dependent increase in the number of annexin V-positive [annexin V(+)]/PI-negative [PI(–)] cells (Fig. 6). To further test the role of PKC{delta} in regulation of apoptosis, CSMCs were treated with PKC{delta} siRNA before 100 nM testosterone treatment. Silencing PKC{delta} expression prevented the increase in annexin V(+)/PI(–) CSMC at both concentrations of testosterone, whereas transfection with control siRNA had no effect on the response to testosterone. These data provide evidence for a key role of PKC{delta} in testosterone-induced CSMC apoptosis.


Figure 6
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  		Fig. 6. Testosterone increases apoptosis of CSMC through PKC-{delta}. CSMC were plated in six-well plates in phenol red-free DMEM medium with 10% charcoal-stripped FBS for 72 h until they reached an ~40–50% confluence. Testosterone (20 and 100 nM) was added to the medium for 24 h. Annexin V FITC binding events were acquired in FACScan. The percentage of annexin V-positive [annexin V(+)], propidium iodide-negative [PI(–)] cells was calculated from fluorescence-1/fluorescence-2 dot plots using quadrant statistics (10,000 cells/experiment). Bar graphs show data (means ± SE) from 4 independent experiments. P < 0.05 vs. nontransfected and control siRNA without testosterone (*) and vs. corresponding nontransfected and control siRNA with testosterone ({dagger}).

 
Testosterone increases caspase 3 activity via PKC{delta}. Activation of caspase 3 is a key event in apoptosis induced by numerous stimuli. To test whether activation of caspase 3 is involved in testosterone-induced CSMC apoptosis, we measured the activity of caspase 3 in the presence and absence of testosterone. Testosterone treatment induced an approximately fourfold increase in caspase 3 activity compared with control CSMC (Fig. 7A), whereas CSMC treated with PKC{delta} siRNA showed an ~75% inhibition of caspase 3 activation in response to testosterone.


Figure 7
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  		Fig. 7. Testosterone increases CSMC caspase 3 active levels through PKC{delta}. A: CSMC were grown until they reached an ~40–50% confluence. Testosterone (20 and 100 nM) was added in the medium for 24 h in phenol red-free DMEM with 10% charcoal-stripped FBS (n = 4). Lysates were assayed for caspase 3 activity using an Enzchek caspase 3 assay kit by Molecular Probes. To evaluate the role of PKC-{delta} on caspase 3 activity, siRNA was used against PKC{delta} for gene silencing. PKC{delta} siRNA treated and untreated with CSMC were incubated for 24 h in the absence or presence of testosterone. CSMC were transfected either with control siRNA (black bars) or PKC-{delta} siRNA (gray bars) and treated with testosterone. Bar graphs show data (means ± SE) from 4 independent experiments. B: to evaluate the significance of increased caspase 3 activity, CSMC were treated with a caspase 3 inhibitor (C3I), Ac-DEVD-CHO (100 µM). CSMC were treated either without (–C3I, black bars) or with (+C3I, gray bars) caspase 3 inhibitor and subsequently treated with testosterone. Later, the cells were subjected to annexin V analysis using FACScan. The percentage of annexin V positive and propidium iodide-negative cells was calculated from fluorescence-1/fluorescence-2 dot plots using quadrant statistics. Bar graphs show data (means ± SE) from 4 independent experiments. *P < 0.05, 0 vs. all. {dagger}P ≤ 0.05, control vs. PKC{delta} siRNA.

 
To evaluate the significance of increased caspase 3 activity in CSMC, we determined the effect of caspase 3 inhibition on testosterone-induced apoptosis. Preincubation with the caspase 3 inhibitor (Ac-DEVD-CHO, 100 µM) substantially reversed the testosterone-induced increase in annexin V(+)/PI(–) cells (Fig. 7B). DeVries et al. (15) have shown that nuclear localization of the 40-kDa catalytic fragment of PKC{delta} is required for apoptosis induced by etoposide. Furthermore, caspase cleavage of PKC{delta} contributes to its pro-apoptotic function by facilitating nuclear import. In CSMCs, testosterone increased the expression of the catalytic fragment of PKC{delta} in a concentration-dependent manner (Fig. 8A), an effect that was completely blocked by the caspase 3 inhibitor Ac-DEVD-CHO (Fig. 8B). Together these data indicate that testosterone-induced increases in the full-length, 80-kDa PKC{delta} increases caspase 3 activity, which in turn cleaves PKC{delta} to produce the catalytic fragment of PKC{delta} to induce apoptosis.


Figure 8
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  		Fig. 8. Testosterone increases the expression of the catalytic fragment of PKC-{delta} through caspase 3. A: densitometric values for effects of testosterone (20 and 100 nM) on the 40-kDa catalytic fragment of PKC-{delta} protein. Bands for each sample were quantified and normalized to the average control value obtained from the same blot. Inset: representative immunoblot probed with anti-PKC{delta} antibody. PKC{delta} positive band appeared at 40 kDa. Testosterone increased 40-kDa PKC{delta} protein levels in a concentration-dependent manner. B: inhibition of caspase 3 activity reversed the effects of testosterone on the expression of the catalytic fragment of PKC{delta} protein. Autoradiograms are representative of 4–5 independent experiments. Values are means ± SE. *P < 0.05 vs. all.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Vascular injury associated with atherosclerosis and coronary interventions stimulate quiescent G0/G1-arrested vascular smooth muscle cells to reenter the cell cycle, replicate DNA, and divide. Proliferating SMCs must pass through several highly regulated cell cycle checkpoints, including G1-to-S and G2-to-M transitions. In this study, we demonstrate that testosterone blocks CSMC cycle progression at the G1-to-S phase transition, attenuates Rb phosphorylation, and upregulates the CDKIs p21cip1 and p27kip1. Furthermore, testosterone-induced G1/Go arrest, downregulation of cyclin D1 and E, and upregulation of p21cip1 were PKC{delta} dependent. These findings present a novel mechanistic basis for the observed beneficial effects of endogenous androgens on vasculoproliferative disease (17, 20, 26, 41, 44, 46).

Phenotypic modulation and proliferation of SMCs are major components of the vasoproliferative response contributing to atherosclerosis and restenosis (47, 52). Balloon angioplasty and stent placement are standard treatment for CAD; however, 30–50% of patients undergoing angioplasty exhibit restenosis and ~20% of those will require additional intervention (47). Cytostatic drugs, such as sirolimus, have emerged clinically as a means to produce cell cycle arrest, inhibit smooth muscle proliferation, and limit restenosis (37). CDKs form activated complexes when bound with cyclins to regulate cell cycle progression. Animal models of vascular injury invariably demonstrate highly coordinated expression of increased cyclins and CDKs and decreased CDKIs, such as p21cip1 and p27kip1, which increase proliferation, migration, and influx of SMCs in the developing lesion (9, 13, 14, 48, 58, 59). Mitogenic activation of cyclin D/CDK4, cyclin D/CDK6, and cyclin E/CDK2 during G1 phase results in Rb protein phosphorylation. Hypophosphorylated Rb functions as a gatekeeper for G1-to-S transition by binding and sequestering E2F, a transcription factor essential for S phase DNA synthesis (51). Cyclin D1 regulates the G1 phase of the cell cycle by binding to and stimulating activities of CDK4 or -6, leading to phosphorylation and inactivation of Rb and subsequent activation of E2F (27). Consistent with this, we show that testosterone decreases the expression of the positive cell cycle regulators cyclin D1 and E and CDK6 activity. Testosterone also reduced levels of pRb, suggesting a possible downregulation of the activity of E2F (12, 23, 32, 36, 40, 43, 59).

We also observed an inhibition of CDK2 activity with testosterone. CDK2 is a serine/threonine kinase whose activity is essential for G1 to S transition during cell activation (10). CDK2 is activated very early after endothelial denudation in a rat carotid model of restenosis (54). Accordingly, CDK2 antisense (40) and CDK2 inhibitors, such as the purine analog CVT-313 (10), inhibit neointimal formation in rat carotid model of restenosis. Consistent with the inhibition of CDK2 activity by testosterone, testosterone also increased levels of the CDKIs p21cip11 and p27kip1, both of which are known to block CDK2 activity (9, 12, 13, 40, 48, 56, 58). Both p27kip1 and p21cip1 regulate cell cycle by binding to and inhibiting activities of cyclin/CDK complexes. Quiescent vascular SMCs express high levels of p21cip1 and p27kip1, whereas mitogenic stimulation decreases expression (48). Studies have shown that adenoviral overexpression of p21cip1 and p27kip1 is effective in preventing neointimal formation (9, 12, 48, 56, 58), whereas p27kip1 null/ApoE null mice demonstrate enhanced atherosclerosis (54).

The anti-proliferative effects of testosterone on CSMC observed in the present study provide a potential mechanism for observations that endogenous testosterone limits vasculoproliferative disease in males through a direct effect on the vascular wall (1, 11). PKC{delta} is known to play an important role in controlling cell proliferation by regulating various cell cycle proteins such as CDK2 kinase activity, p21cip1, and cyclin D1, which have been shown through knockout and overexpression studies to reduce neointimal formation both in vivo and in vitro (9, 10, 13, 36, 48, 50, 58). Overexpression of PKC{delta} in rat aortic SMCs inhibited growth and proliferation, decreased thymidine incorporation, induced G0/G1 arrest, reduced cyclin D1 and E protein, and increased p27kip1 (25). This cytostatic profile produced by PKC{delta} is nearly identical to that produced in CSMC by testosterone. Specifically, testosterone inhibited CSMC proliferation, decreased thymidine incorporation, induced G0/G1 arrest, reduced cyclin D1 and E protein levels, decreased CDK2 and CDK6 activities, and increased both p21cip1 and p27kip1. Furthermore, knockdown of PKC{delta} using siRNA demonstrated that the effects of testosterone on cyclin D1, cyclin E, and p21cip1 were dependent on PKC{delta}. The lack of an effect of PKC{delta} knockdown on p27kip1 upregulation by testosterone is not consistent with PKC{delta} overexpression (25), suggesting that testosterone-induced upregulation of p27kip1 is not dependent on PKC{delta}. Thus testosterone appears to exert some PKC{delta}-independent effects that may account for the residual inhibition of CSMC proliferation in the presence of PKC{delta} siRNA.

The past decade has seen an increase in the number of studies examining the role of SMC apoptosis during arteriosclerosis and restenosis (29, 31). The current study provides the first evidence for a PKC{delta}-dependent mechanism whereby physiological levels of testosterone, i.e., 20 nM (8, 53), increase CSMC apoptosis as evidenced by annexin V staining and catalytic PKC{delta} formation. The present study provides several insights into the underlying mechanism of testosterone-mediated, PKC{delta}-dependent apoptosis. Our results using PKC{delta} siRNA demonstrate that testosterone-induced apoptosis is largely dependent upon PKC{delta}. Interestingly, PKC{delta} null mice exhibit decreased SMC apoptosis and increased neointimal lesions induced by vein grafts (34), supporting a crucial role of PKC{delta} in mediating SMC apoptosis and regulating lesion development. Nuclear translocation of PKC{delta} is augmented by caspase cleavage of the full-length 80-kDa PKC{delta} to form the 40-kDa catalytic fragment (15). Mutation of the caspase 3 cleavage site severely inhibited the ability of PKC{delta} to translocate to the nucleus. Similarly, in this study, inhibition of caspase 3 inhibited both testosterone-induced apoptosis and PKC{delta} cleavage. Furthermore, caspase 3 activation by testosterone was dependent upon PKC{delta}, as demonstrated by PKC{delta} siRNA. Taken together, our studies indicate that caspase 3 cleavage of PKC{delta} is an important component of the apoptotic response induced by testosterone, likely allowing for nuclear accumulation of calalytic PKC{delta} during apoptosis. In context with our previous findings that testosterone and DHT increase PKC{delta} expression and activity in CSMCs (35), the current findings support a model whereby testosterone-dependent increases in full-length PKC{delta} levels trigger increased SMC apoptosis via an increase in caspase 3-mediated production of the 40-kDa catalytic fragment of PKC{delta}. These findings represent the first mechanistic linking of testosterone and PKC{delta} to CSMC apoptosis.

Peripheral tissues express aromatase and 5{alpha}-reductase, which convert testosterone to DHT or 17beta-estradiol, respectively, allowing tissue-specific control over the immediate hormonal milieu. Local conversion of testosterone to estrogen via aromatase has been proposed to mediate testosterone effects in the brain (3), vascular SMCs, and ovary (57). Porcine coronary smooth muscle and endothelium express both androgen and estrogen receptors (8, 42). Our finding in this study that DHT inhibited [3H]thymidine incorporation similar to testosterone provides evidence that aromatization of testosterone to estrogen is not necessary for PKC{delta}-mediated inhibition of CSMC proliferation by testosterone.

In conclusion, data presented here support a model whereby testosterone-dependent increases in active PKC{delta} protein levels are associated with inhibition of SMC proliferation and stimulation of apoptosis. We have previously shown in the swine model that endogenous testosterone is a potent stimulator of PKC{delta} protein levels and kinase activity in coronary smooth muscle (35). Thus these findings represent the first mechanistic link between testosterone, PKC{delta} activity, and regulation of CSMC proliferation and apoptosis. The anti-proliferative, pro-apoptotic actions of testosterone on CSMC support the concept that maintaining adequate levels of testosterone may present a therapeutic strategy against vasculoproliferative diseases such as CAD and/or restenosis in males.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-079934 HL-071574, by the National Aeronautics and Space Administration (D. K. Bowles), and by American Heart Association Predoctoral Fellowship 0515635Z (K. K. Maddali).


   ACKNOWLEDGMENTS
 
We thank Dr. Chadda Reddy for valuable input in this study and Dr. Venkataseshu Ganjam for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Bowles, E102 Veterinary Medicine, Univ. of Missouri, Columbia, MO 65211 (e-mail: BowlesD{at}missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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