PKCδ Mediates Testosterone-induced Increases in Coronary Smooth Muscle Cav1.2*

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Cav1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase C δ (PKCδ) expression and whether PKCδ plays a role in coronary Cav1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nm) for 18 h at 37 °C in a humidified chamber. For sex and endogenous testosterone-dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an ∼2-3-fold increase in PKCδ protein levels, ∼1.5-2-fold increase in PKCδ kinase activity, and localization of PKCδ toward the plasma membrane and nuclear envelope. PKCδ protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCδ protein levels, an effect partially (∼45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCδ inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Cav1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Cav1.2 protein levels. PKCδ short interference RNA completely blocked T-induced increases in Cav1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCδ protein and activity in males and 2) T increases Cav1.2 protein expression in a PKCδ-dependent manner.

Coronary heart disease is a major cause of global mortality and represents an underlying cause for most heart attacks and sudden death (1). Men 30 -50 years of age have an increased incidence of coronary heart disease compared with women of similar age (2-4), a sex difference that led many to the conclusion that testosterone increases the risk of coronary heart disease in men. However, recent clinical studies have failed to support a detrimental effect of testosterone on coronary heart disease (5)(6)(7). On the contrary, low testosterone concentrations in men are associated with a higher risk of atherosclerosis (7). Men with low testosterone levels are often more obese, hypertensive, and have increased blood glucose and serum cholesterol levels and increased carotid artery atherosclerosis with diabetes (5,7). Additionally, Dunajaska et al. (6) have shown that low levels of total testosterone, testosterone/estradiol ratio, and free androgen index are associated with coronary artery disease in men. These findings underscore the need to fully understand the effects of testosterone on coronary vascular wall biology.
Sex hormones exert multiple and diverse effects on the vascular wall. Both endothelial and vascular smooth muscle cells express estrogen (8) and androgen receptors (9,10). We recently provided the first evidence for an increased L-type voltage-gated calcium channel (Ca v 1.2) current in coronary artery smooth muscle of male swine compared with females (11). Subsequently, we demonstrated that coronary Ca v 1.2 expression and activity are stimulated by endogenous testosterone in males (12). In vitro, both testosterone and non-aromatizable androgen, dihydrotestosterone (DHT), 2 increased Ca v 1.2 protein levels (12). Thus, endogenous testosterone increases Ca v 1.2 expression in porcine coronary smooth muscle cells. However, the mechanisms by which androgens elevate Ca v 1.2 protein levels in males are poorly understood.
Many aspects of vascular smooth muscle biology, including contraction, differentiation, proliferation, apoptosis, and myogenic responses, share common signaling pathways involving the activation of protein kinase C (PKC) (13,14). For example, acute activation of PKC with phorbol esters increases Ca 2ϩ influx in vascular smooth muscle, purportedly via PKC-dependent activation of voltage-gated Ca 2ϩ channels (15,16). Kanashiro et al. (17) showed an increased expression and activity of PKC␣, -␦, and -in aortas from intact males and ovariectomized females compared with intact females, revealing an isoform-specific effect of sex hormones, specifically estrogen, to PKC expression and activity. We previously reported a 4-fold greater expression of PKC␦ protein levels in male coronary arteries compared with females, a sex difference that was eliminated by castration of males (18), suggesting increased PKC␦ levels in males might be driven by male sex hormones, e.g. testosterone.
Despite evidence for an important regulatory role of PKC in vasoreactivity and vascular disease progression, to our knowledge no information exists regarding androgen effects on PKC␦ expression and its role in coronary Ca v 1.2 protein expression. In the present study, we therefore compared expression, activity, and subcellular distribution of PKC␦ in coronary smooth muscle following testosterone (T) stimulation and, in addition, tested the hypothesis that testosterone-induced increases in Ca v 1.2 were PKC␦ dependent. The results demonstrate that androgens increase both * This study was supported by NHLBI, National Institutes of Health Grant HL071574 and by the National Aeronautics and Space Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PKC␦ activity and expression, which serve as a mandatory intermediate for testosterone-induced increases in Ca v 1.2 protein levels.

MATERIALS AND METHODS
Animals-Sexually mature male and female Yucatan swine were obtained from the breeder (Sinclair Research Farm; Columbia, MO) and housed in pens at the College of Veterinary Medicine. Animal protocols were approved by the University of Missouri Animal Care and Use Committee in accordance with the "Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training" as described previously (12).
Castration and Hormone Replacement-Data for PKC␦ in swine with altered hormone status were obtained from coronary arteries of swine utilized in a previous study (12). As described previously (12), castration (orchiectomy) and hormone replacement were performed by the Swine Hormone Core within the National Center for Gender Physiology. One week after arrival, sexually mature animals (6 -7 months of age) were castrated with aseptic techniques under sedation with xylazine (15 mg/kg im) and ketamine (2.5 mg/kg intramuscular) and maintained under anesthesia with 1.5-2.0% isoflurane. Males were castrated (CM) via an incision made through the scrotum over each testicle (intact males; IM) and subsequently randomized to receive testosterone replacement (CMT; 10 mg/day; Androgel, Solvay Pharmaceuticals) or vehicle. Testosterone replacement occurred at the time of castration to avoid disruption of hormonal influence. Females remained gonadally intact. Animals were euthanized for study 5-6 weeks after surgery.
Isolation of Coronary Arteries-Miniature swine were anesthetized with ketamine (35 mg/kg), rompun (2.25 mg/kg), and pentothal sodium (10 mg/kg) followed by administration of heparin (1000 units/kg). Swine were euthanized by removal of the heart, and the heart was placed in 4°C PSS. The right coronary artery (RCA) was isolated, cleaned of fat and connective tissue, and placed in low Ca 2ϩ physiological saline solution containing 20 mM HEPES at 4°C.
Coronary Artery Culture and Treatment-An intact coronary artery tissue culture model was used to study sex hormone effects on coronary artery smooth muscle in vitro as modified from Hill et al. (19) and Maddali et al. (20). RCA (intact vessels) were cut into 1-cm rings and incubated for 18 h in the presence and absence of testosterone (0 and 100 nM) or DHT (10 and 100 nM) in serum-free, phenol-free RPMI 1640 medium (SFM) with L-glutamine at 37°C in a 5% CO 2 , humidified incubator.
Coronary Smooth Muscle Cell Cultures and Treatment-Primary cell cultures (passages 3-6) of medial CSMC were obtained from the RCA. Post-confluent, 5-day serum-restricted cells were treated with varying doses of testosterone (10 and 100 nM) or DHT (10 and 100 nM) for 18 h in SFM at 37°C in a humidified chamber.
Kinase Activity Assays-Kinase activity levels were determined using standard PKC kinase activity assays as described (17). Intact vessels (RCA) and CSMC treated with T or DHT were homogenized or lysed, respectively, in phosphorylation lysis buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% ␤-mercaptoethanol, 1 mM phenyl-methylsulfonyl fluoride, 2 mM leupeptin, 1 mM pepstatin A, 10% v/v glycerol, and 0.25% Triton X-100). Homogenates were immunoprecipitated with an antibody against PKC␦ using protein G-agarose (Amersham Biosciences). The immunocomplexes were washed three times with phosphorylation lysis buffer and two times with kinase buffer (25 mM Tris-HCl (pH 7.4), 5 mM Mgcl 2 , 0.5 mM EGTA, 1 mM dithiothreitol, 20 mg of phosphatidylserine, and 20 mM ATP, 25 mM Mgcl 2 , 25 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate) and were resuspended in 30 l of kinase buffer containing 5 mg of histone H1, and then 20 -30 Ci of [␥-32 P] ATP was added. The reaction was incubated for 15-30 min at room temperature and was terminated by the addition of SDS-sample buffer. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography.
Immunocytofluorescence-Freshly dispersed coronary smooth muscle cells were obtained enzymatically from RCA segments as previously described (20). Cells were fixed on polylysine-coated coverslips (BD Biosciences) in 4% paraformaldehyde, permeabilized in phosphatebuffered saline plus 0.1% Triton X-100, and incubated in phosphatebuffered saline plus 5% bovine serum albumin and 5% goat serum for 30 min. Cells were washed and incubated overnight at 4°C in primary rabbit polyclonal antibodies for PKC␦ (1:1000) and tagged with goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Nuclei were visualized with the nuclear dye, propidium iodide (5 ϫ 10 Ϫ5 M, 10 min; Molecular Probes). Images were obtained using a ϫ60 water immersion objective (1.2 NA) on an Olympus IX-70 (Tokyo, Japan) inverted microscope coupled with a Bio-Rad Radiance-2000 confocal system. Optical sections (10 -15) were obtained at 0.7-m intervals along the z-axis. Specificity of secondary antibody was verified in all experiments by addition of secondary antibody in the absence of primary antibody, which showed no fluorescent signal (not shown). Cells prepared from five animals were dispersed onto eight slides. Approximately 40 -50 cells were imaged for PKC␦ isoform, and representative images were presented.
PKC␦ siRNA Protocol-Transfection was performed according to the manufacturer's protocol (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, 5 l of Lipofectamine 2000 (Invitrogen) and 20 pM PKC␦ siRNA or control siRNA pool (Santa Cruz Biotechnology) were diluted in 250 l of serum-free phenol red-free Dulbecco's modified Eagle's medium and incubated at room temperature for not more than 5 min. The diluted siRNA pools were then mixed with the diluted Lipofectamine 2000 and incubated at room temperature for 20 min. For transfection, post-confluent cells were subcultured at 90% confluence and serum starved for 2 days. Cells were then incubated at 37°C for 6 h with siRNA-Lipofectamine 2000 complexes. After the transfection period, the medium was removed, the wells were washed twice with SFM, and 2 ml of fresh SFM was added. The cultures were then allowed to recover for 48 h with SFM changes every 24 h. At the end of the 48-h recovery period, the cells in 1-2 wells from each siRNA group (control and PKC␦) were harvested and cell numbers were evaluated. The remaining wells in each group were either treated with ethanol (vehicle controls) or T (100 nM). The total treatment time was 18 h. The cells in each group were collected, pelleted, and used for immunoblots analysis. All the siRNA experiments were conducted in serum-free medium containing no antimicrobials.
RPMI 1640 medium, phosphate-buffered saline, goat serum, and rabbit serum were obtained from Invitrogen. Antibodies to PKC isoforms were obtained from Santa Cruz Biotechnology. Antibody to Ca v 1.2 was purchased from Alomone Labs (Jerusalem, Israel). Molecular mass markers and Tris-HCl gels were purchased from Bio-Rad. Horseradish peroxidase-conjugated goat anti-rabbit IgG and ECL Western blotting detection reagents were obtained from Amersham Biosciences.
Statistical Analysis-All values are expressed as mean Ϯ S.E. Comparisons among treatment groups were made using analysis of variance with Bonferroni post hoc analyses when indicated. A p value Յ0.05 was set as the criterion for significance in all comparisons.

RESULTS
To determine whether the sex differences in PKC␦ expression were driven by testosterone, we examined the influence of sex on PKC␦ protein in an in vivo model. As we have previously demonstrated (18), males were found to have greater PKC␦ compared with intact females (Fig. 1). Castration markedly reduced PKC␦ protein levels in males, whereas testosterone replacement partially (ϳ45%) prevented the loss of protein due to castration (Fig. 1). Thus, endogenous testosterone increases coronary PKC␦ protein and likely contributes to greater PKC␦ protein levels in coronary arteries of males.
To further investigate the mechanism of testosterone stimulation of PKC␦ protein expression (Fig. 1), we determined the effects of testosterone on PKC␦ protein levels in an in vitro cell culture model and organ culture model (intact RCA). For this, PKC␦ expression was determined in homogenates obtained from intact RCA and post-confluent, 5-day serum-restricted coronary smooth muscle cells incubated with testosterone or dihydrotestosterone. In intact vessels, both testosterone and dihydrotestosterone produced similar, concentration-dependent increases in PKC␦ protein with a maximal response of ϳ2.5and ϳ3.5fold, respectively, at 100 nM (Fig. 2, A and C). We observed a similar response in CSMC (Fig. 2, B and D), whereby PKC␦ protein levels increased ϳ2.3and ϳ3.6-fold following testosterone and dihydrotestosterone treatment, respectively. Together with intact vessel data, these results confirm the up-regulation of PKC␦ protein levels by testosterone. In vivo, testosterone can be converted to either estrogen or dihydrotestosterone by aromatase or 5-␣ reductase, respectively. Dihydrotestosterone is a non-aromatizable androgen that acts through binding to the androgen receptor. The similar effects of testosterone and DHT on PKC␦ protein levels provide strong evidence that testosterone conversion to estrogen by aromatization is not necessary for these observed effects of testosterone on PKC␦.
To determine whether testosterone affects PKC␦ kinase activity,  PKC␦ protein was immunoprecipitated from homogenates of intact vessels and cultured CSMC treated with testosterone (10 or 100 nM) or DHT (10 or 100 nM). Testosterone treatment significantly elevated PKC␦ kinase levels in both intact vessels and primary CSMC in a concentration-dependent manner (Fig. 3, A and B). Densitometric analysis indicated ϳ1.5and ϳ3-fold increases in total PKC␦ activity with 10 and 100 nM testosterone, respectively. DHT also increased PKC␦ kinase activity in a concentration-dependent manner (ϳ5-and ϳ7-fold for 10 and 100 nM, respectively) in both intact vessels and CSMC (Fig. 3, A and  B). These data provide evidence that a non-aromatizable androgen, DHT, mimics the effects of testosterone on PKC␦ kinase activity.
To evaluate the subcellular distribution of PKC␦ in response to testosterone, CSMC were dispersed from testosterone-treated RCA and subcellular distribution examined by confocal immunocytochemistry. In untreated cells, PKC␦ was diffusely distributed throughout the cytosol (Fig. 3C, left). Conversely, cells treated with 100 nM testosterone showed a localization of PKC␦ toward plasma membrane and a perinuclear, reticular pattern that extended into the central cytosol (Fig. 3C,  right). These data demonstrate a specific subcellular distribution of PKC␦ in response to testosterone, suggesting a role for PKC␦ in membrane and nuclear signaling.
We have previously demonstrated that testosterone drives Ca v 1.2 mRNA and protein expression in coronary smooth muscle (12). The finding in the present study that testosterone also increases coronary smooth muscle PKC␦ protein levels is consistent with previous sex differences in coronary PKC␦ protein levels (18) and led us to determine the contribution of PKC␦ in testosterone-mediated effects on Ca v 1.2 protein expression. RCA segments and primary CSMC were pretreated with chelerythrine (10 M), a general inhibitor of PKC, Go6976 (5 M), a conventional PKC isoform inhibitor, or rottlerin (5 M), a putative PKC␦-selective inhibitor, and incubated for 18 h in the presence/ab-sence of testosterone (100 nM). Both chelerythrine and rottlerin inhibited testosterone-induced Ca v 1.2 protein levels (Fig. 4). In contrast, Go6976 had no effect on T-induced Ca v 1.2 protein levels. These data demonstrate that testosterone stimulation of Ca v 1.2 protein expression in coronary smooth muscle is mediated by PKC␦ and/or novel PKC isoforms with no apparent contribution from conventional PKC isoforms.
To further test the hypothesis that PKC␦ is the specific isoform involved in testosterone up-regulation of Ca v 1.2, suppression of PKC␦ protein was accomplished using siRNA (Fig. 5). Successful RNA silencing was demonstrated by knockdown of PKC␦ protein by ϳ90% (Fig. 5). To demonstrate specificity, siRNA-treated CSMC were probed for additional PKC isoforms, i.e. PKC␣, -⑀, and - (Fig. 5). PKC␦ siRNA essentially abolished PKC␦ protein levels without altering the expression of PKC␣, -⑀, and -in CSMC, providing evidence for specific downregulation of PKC␦. Importantly, PKC␦ siRNA completely blocked the stimulatory effect of testosterone on Ca v 1.2 protein expression (Fig. 6). These data demonstrate that testosterone increases Ca v 1.2 protein expression through a PKC␦-dependent mechanism.

DISCUSSION
The present study provides several novel insights into testosterone regulation of coronary arteries, including that 1) stimulation of coronary PKC␦ by endogenous testosterone in vivo, 2) stimulation of coronary smooth muscle PKC␦ expression and activity by both testosterone and dihydrotestosterone in vitro, and 3) testosterone-induced increases in Ca v 1.2 expression in coronary smooth muscle are dependent upon increases in PKC␦. Specifically, testosterone increased PKC␦ protein levels and activity and altered the subcellular localization of PKC␦. In addition, these studies provide a mechanism for our previous finding that endogenous testosterone increases in Ca v 1.2 protein levels, mRNA expression, and activity in male  DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 coronary arteries (12). Furthermore, these findings demonstrate that testosterone, within the physiological range of total serum testosterone in humans (21), is a selective and potent regulator of PKC␦ protein levels in coronary artery smooth muscle.

Testosterone, Ca v 1.2, and Coronary PKC␦
Recent studies have concluded that sex differences in PKC contribute to differences in vascular smooth muscle reactivity (17,18). In the rat aorta, estrogen has been identified as the primary mediator of sex differences in vascular reactivity and PKC␣, -␦, and -expres-sion, with no effect of testosterone (17,22). However, in our porcine model, castration reduced, and testosterone replacement abrogated, coronary PKC␦ protein levels. The efficacy of castration and testosterone replacement in these animals has been previously published (12); thus coronary PKC␦ levels directly correlate with endogenous testosterone levels. It is of interest to note that testosterone replacement did not completely prevent the loss of PKC␦ despite maintenance of total serum testosterone levels similar to those in intact controls (12). This could be due to our inability to adequately mimic the episodic secretion profile of testosterone by our replacement regimen or the contribution of another, as yet unidentified, gonadal androgen to PKC␦ regulation. Additional studies will be needed to completely describe the regulation of PKC␦ by male sex hormones. However, our in vitro findings with testosterone and DHT clearly support a prominent role of androgens in stimulating coronary smooth muscle PKC␦ expression and activity. Together these studies demonstrate potential species-and vascular bed-specific effects of sex hormones that should be considered when interpreting sex comparisons. L-type, voltage-gated Ca 2ϩ calcium channels are heteromeric complexes minimally composed of three protein subunits, ␣ 1 , ␣ 2 /␦, and ␤. The ␣ 1 subunit (Ca v 1.2) forms the channel pore and contains the binding sites for dihydropyridine antagonists. L-type Ca 2ϩ channels mediate calcium entry into smooth muscle cells and play a central role not only in excitation-contraction coupling but also in gene expression and differentiation (23,24). Our previous studies report a stimulatory effect of testosterone on Ca v 1.2 mRNA and protein levels (11,12), suggesting a transcriptional and translational stimulation of the Ca v 1.2 gene by testosterone. Accordingly, Liu et al. (25) showed a hormone-responsive element in 5Ј flanking region activated by testosterone in cardiac and smooth muscle cells. The present study involving various PKC isoform inhibitors and siRNA clearly shows a regulation of Ca V 1.2 protein levels through the novel isoform PKC␦. Rottlerin, a putative PKC␦ inhibitor, and chelerythrine, a generalized PKC inhibitor, blocked the testosterone-induced increase in Ca v 1.2 protein levels. In contrast, Go6976, a conventional PKC isoform inhibitor, had no effect on testosterone stimulation of Ca v 1.2 protein. Furthermore, failure of testosterone to increase Ca v 1.2 protein levels in the presence of PKC␦ siRNA con-   firmed the mandatory role of PKC␦ in testosterone-induced Ca v 1.2 protein up-regulation.
Although the present study is the first to report PKC␦-dependent up-regulation of Ca v 1.2 protein in smooth muscle, these findings are consistent with PKC␦-dependent up-regulation of Ca v 1.2 protein by ethanol in PC12 cells (26,27). Whether PKC␦ acts via a translational or post-translational mechanism is unknown. The Ca v 1.2 promoter region contains numerous cis-acting response elements, including Nkx2.5, CRE-BP, HRE, C/EBPb and AP-1 (25), while PKC␦ has been shown to stimulate AP-1/Jun-responsive genes (28). However, in PC12 cells, upregulation of Ca v 1.2 protein, but not mRNA, was PKC␦ dependent (26), indicating a post-translational regulation of Ca v 1.2 by PKC␦ in these cells. We previously reported that testosterone increases both mRNA and protein levels of Ca V 1.2 in coronary smooth muscle (12); however, it remains unknown whether the obligatory role of PKC␦ in testosterone-induced increases in Ca v 1.2 protein is post-translational or via increased Ca v 1.2 gene expression.
Most peripheral tissues express aromatase and 5␣-reductase, which convert testosterone to 17␤-estradiol or dihydrotestosterone, 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 (29), vascular smooth muscle cells, and ovary (30). Porcine coronary smooth muscle and endothelium express both androgen and estrogen receptors (12,31). Our finding in this study that DHT produced up-regulation of PKC␦ protein and activity levels similar to testosterone demonstrates that aromatization of testosterone to estrogen (31,33) is not necessary for up-regulation of PKC␦ protein levels, activity, and PKC␦-mediated Ca v 1.2 protein levels by testosterone.
The up-regulation of PKC␦ by testosterone in the present study likely contributes to increased Ca V 1.2 protein and activity levels in coronary arteries of males compared with females (11,12), providing a novel and important mechanism for sex differences in coronary pathophysiology. For example, testosterone-mediated increases in coronary smooth muscle PKC␦ may represent a primary mechanism underlying recent clinical evidence that physiological levels of testosterone protect against atherosclerosis in males (5-7). PKC␦-deficient mice show enhanced atherosclerotic lesion development in vein grafts, primarily because of enhanced smooth muscle cell infiltration (34), consistent with anti-proliferative, pro-apoptotic effects of PKC␦. Therapeutically, maintenance of normal endogenous testosterone in males may retard the progression of atherosclerotic lesion development by maintaining optimal levels of PKC␦ in coronary smooth muscle.
In conclusion, the present study clearly demonstrates that testosterone is a primary modulator of PKC␦ protein activity and PKC␦-dependent increases in Ca v 1.2 protein expression in coronary smooth muscle. These studies provide the first mechanistic data linking testosterone regulation of Ca v 1.2 protein levels to PKC␦. Recent studies have demonstrated that both Ca v 1.2 and PKC␦ control smooth muscle cell contraction, differentiation, and proliferation (23,32). The present study provides the foundation for future studies to determine the effect of testosterone on coronary smooth muscle phenotype modulation and, ultimately, whether these factors contribute to the apparent salutatory effect of testosterone on the development of coronary artery disease.