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Endocrinology Vol. 150, No. 1 396-403
Copyright © 2009 by The Endocrine Society

Cyclin-Dependent Kinase 5 Regulates Steroidogenic Acute Regulatory Protein and Androgen Production in Mouse Leydig Cells

Ho Lin, Mei-Chih Chen and Chien-Te Ku

Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan

Address all correspondence and requests for reprints to: Ho Lin, Ph.D., Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan. E-mail: hlin{at}dragon.nchu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclin-dependent kinase 5 (CDK) 5 IS A UNIQUE member of a small serine/threonine Cdk family. Although most Cdk proteins are involved in cell cycle regulation, Cdk5 is primarily involved in neuronal development and degeneration (1). In the central nervous system (CNS), Cdk5 is involved in neuronal cytoskeleton regulation, axon guidance, membrane transport, synaptic function, and dopamine signaling as well as drug addiction (2). Like other Cdk proteins, Cdk5 alone shows no kinase activity and requires the association with the regulatory partner p35 to activate and maintain neuronal processes (3). Our recent results indicate that Cdk5 plays decisive roles in both neurodegeneration and cancer biology (4, 5). Expressions of Cdk5/p35 proteins have also been described in several reproductive cell types, including Leydig cells, Sertoli cells, and prostate cells (6, 7, 8). In mouse Leydig cell line (TM3), Cdk5 protein levels and kinase activity were significantly elevated that were grown in the presence of epidermal growth factor or LH and were associated with an increase in testosterone production (6). Our previous results show that Cdk5 involves drug-induced apoptosis of prostate cancer cells that respond to androgen secreted by Leydig cells (9). Therefore, it was of great interest to investigate whether the Cdk5/p35 kinase modulates androgen production in Leydig cells, in which Cdk5/p35 are known to exert direct or indirect influences on androgen-dependent functions and diseases, such as prostate cancer.

Leydig cells are distributed in the interstitium of the testis and are known as the main source of androgen production in mammals (10). The steroidogenic function of Leydig cells is primarily regulated by LH/human chorionic gonadotrophin (hCG) as well as multiple signaling pathways (11, 12, 13). The steroidogenic potential of Leydig cells under LH/hCG controls can be modulated by both steroidogenic enzymes and steroidogenic acute regulatory (StAR) protein, which is important in cholesterol transport processes from cytoplasm into mitochondria (14). The StAR protein is critical in early regulation of steroidogenesis and has been investigated in several in vitro and clinical studies (15, 16, 17, 18). Whereas expression of StAR protein is predominantly regulated by cAMP-dependent mechanisms downstream to the LH/hCG receptor, several other factors and signaling pathways are also known to play important regulatory roles (16, 19). Although substantial evidence indicates mechanisms of StAR expression regulation (20), it remains unclear whether stability of StAR protein might be regulated in steroidogenesis. Because StAR protein induces rapid effects, it is extremely important to describe and understand StAR protein accumulation in addition to regulation of expression.

In the current study, we show that Cdk5/p35 is important to hCG-dependent androgen production in both a mouse Leydig cell line and in primary cells due to the contribution of StAR protein regulation. Thus, Cdk5 appears to be a novel player in male reproduction and a potential treatment target in androgen-related diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection
Mouse TM3 cells were provided by Professor Paulus S. Wang (Department of Physiology, National Yang Ming University, Taiwan). TM3 cells were cultured in Ham F12+DMEM medium (Sigma, St. Louis, MO) plus 5% horse serum (Hyclone, Logan, UT), 5% fetal bovine serum (Life Technologies, Inc., Grand Island, NY), and penicillin/streptomycin (Sigma) at 37 C in a humidified atmosphere at 5% CO2. Male mouse of C57BL/6 strain (2–3 months of age) were housed in a temperature-controlled room (22 ± 1 C) with 14 h of artificial illumination daily (6–20 h) and given food and water ad libitum before isolation of Leydig cells. Isolation and purification of Leydig cells from mouse were carried out using previously described procedures (21, 22, 23). Decapsulated interstitial cells were dispersed by collagenase (0.2%, 20 min, 34 C; Sigma), filtered through sterile nylon gauze (0.5–0.8 mm mesh), and washed with M199 medium (Sigma) to remove the collagenase. Cells were then purified using continuous Percoll density gradient (range 1.01–1.126 kg/liter) centrifugation. During centrifugation, cell types partitioning at approximately 1.07 kg/liter of Percoll were collected and washed, and 80–90% of the cells were Leydig cells as determined histochemically by 3β-hydroxysteroid dehydrogenase staining. Cells were subcultured in DMEM/F12 medium (Sigma) containing 10% fetal bovine serum (Life Technologies) and antibiotics (penicillin/streptomycin) and used for experiments after 48 h. All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University (Taiwan). Mouse small interfering RNA (siRNA)-cdk5 (M-040544-00) and a nonspecific control siRNA were purchased from Dharmacon (SMARTpool; Lafayette, CO). Mouse cdk5 expression plasmids were constructed through RT-PCR amplifications of the mouse cdk5 coding sequences and inserted into the pcDNA4 vector (Invitrogen, Carlsbad, CA) by thymidine/adenosine overhanging cloning. Introductions of siRNA and plasmids into TM3 cells were performed by using Lipofectamine 2000 (Invitrogen) with 50 pmol siRNA/105 cells.

Western blotting and immunoprecipitation analysis
Cell lysate was isolated for immunoprecipitation in either lysis buffer [20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 137 mM NaCl, 50 µM EDTA, protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride] or extract buffer (100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). Immunoprecipitates were collected by binding to protein G Plus/protein A-Agarose beads (IP-05; Merck, Darmstadt, Germany). Proteins were subsequently analyzed by direct Western blotting (30 µg/lane) or blotting after immunoprecipitation (1–2 mg/immunoprecipitation). The antibodies included anti-Cdk5 antibody (05–364; Upstate, Lake Placid, NY), anti-p35 antibody (sc-820; Santa Cruz Biotechnology, Santa Cruz, CA), antiactin antibody (MAB1501; Chemicon, Temecula, CA), anti-StAR antibodies [a gift from Dr. Walter L. Miller, University of California, San Francisco, San Francisco, CA (24); FL-285, Santa Cruz; K-20, Santa Cruz], anti-phosphoserine antibody (AB1603; Chemicon), and peroxidase-conjugated antimouse, antirabbit, or antigoat antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA). An enhanced chemiluminescence detection reagent (PerkinElmer, Shelton, CT) was used to visualize immunoreactive proteins on polyvinyl difluoride membranes.

In vitro Cdk5 kinase assay
The kinase assay was performed by washing immunoprecipitates three times with kinase reaction buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, and 1 mM dithiothreitol]. The protein G Plus/protein A-agarose beads with target proteins were incubated in kinase reaction buffer containing 2 µg substrate (histone H1, 14–155; Upstate) and ATP in a final volume of 40 µl at 30 C for 30 min. The signals of phospho-histone H1 were identified by immunoblotting with the anti-phosphohistone H1 antibody (Upstate).

Immunocytochemistry
TM3 cells cultured on coverslips were washed twice by PBS and fixed for 10 min in 4% paraformaldehyde and 2% sucrose in PBS at room temperature. After fixation, cells were permeabilized with buffer containing 0.3% Triton X-100 and 3% BSA in PBS for 2 min at room temperature. PBS wash and subsequent block in 3% BSA-PBS were each performed for 15 min at room temperature. Antibodies used in immunostaining included anti-Cdk5 (Upstate), anti-p35 (Santa Cruz), and anti-StAR (FL-285; Santa Cruz). Images were scanned using Leica confocal microscopy (Taichung Veteran General Hospital, Taiwan).

Measurement of testosterone
Media and cell lysates of TM3 cells were extracted by adding 1 ml diethyl ether. The two layers were allowed to separate and the ether layer was collected by freezing the lower aqueous layer in an acetone/ice bath and decanting the upper layer. The ether extracts were evaporated completely. The resulting pellets were dissolved in 1 ml of assay buffer [50 mM Tris buffer (pH 8.0) containing 0.1% (wt/vol) gelatin]. Each sample was assayed for testosterone content by ELISA (KAP1701; Biosource, Camarillo, CA) according to the manufacturer’s protocols (6, 25).

RT-PCR
Total RNA was isolated from TM3 cells using the spin column RNA miniprep kit according to the manufacturer’s instructions (SK361, Genemark, Taiwan). Reverse transcribed-PCR (RT-PCR) was performed using the One-Step RT-PCR kit (RP01; Genemark, Taipei, Taiwan). StAR-specific primers were synthesized according to previous report (20) (sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3'). The RT-PCRs were performed using 10 pmol of each primer and the following program: 30 min at 50 C and 2 min at 94 C, followed by 18 cycles of denaturing at 94 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 1 min, ending with a final extension at 72 C for 7 min. The amplified PCR products were separated by electrophoresis on a 0.8% agarose gel and visualized by ethidium bromide staining. A 1-kb DNA ladder (Genemark) was used as size marker.

Statistics
All values were given as the mean ± SEM. In all cases, means were analyzed using Student’s t test. A difference between two means was considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hCG regulates Cdk5/p35 protein expression and kinase activity in dose- and time-dependent manners in mouse Leydig cells
Different hCG dosages (0–0.2 IU/ml) and treatment durations (0–48 h) were administered to the mouse Leydig cell line (TM3), and Cdk5/p35 protein expression and kinase activity were analyzed in low serum conditions (1% horse serum + 1% fetal bovine serum). Our results indicated that protein expression and kinase activity of Cdk5 and p35 were enhanced by hCG treatments in both a dose- and time-dependent manner (Fig. 1Go). In addition to the results from the TM3 cell line, we found that protein expression and kinase activity of Cdk5/p35 were regulated by hCG (0.1 IU/ml in low serum condition, 0–24 h) in mouse primary Leydig cells (Fig. 2Go). Because treatment with hCG at 0.1 IU/ml for 24 h induced the greatest increase in protein expression, these treatment conditions in low serum environments were used throughout the rest of study. In addition, the evidence indicated that proliferation of TM3 cells was affected by LH treatment (6). Our data showed that no significant influence of proliferation was found between control group and hCG-treated groups (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).


Figure 1
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  		FIG. 1. hCG treatments increase both Cdk5/p35 protein expression and kinase activity in TM3 cells in dose- and time-dependent manners. TM3 cells were treated with different dosages of hCG (0–0.2 IU/ml) for different time intervals under low serum conditions (1% horse serum plus 1% fetal bovine serum). Protein expression and Cdk5 kinase activity were measured by Western blotting and in vitro kinase assay, respectively. Actin represents the internal control for protein expression. Quantitative results of hCG-dependent effects on protein expression and kinase activity (n = 3). A, The effects of hCG in a dose-dependent manner. B, The effects of hCG in a time-dependent manner. Control value was for 1. **, P < 0.01 vs. control group.

 

Figure 2
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  		FIG. 2. hCG treatments increase both Cdk5/p35 protein expression and kinase activity in mouse primary Leydig cells in a time-dependent manner. Primary Leydig cells were treated with hCG (0.1 IU/ml) for different time intervals (0, 12, 24 h) under low serum conditions (1% horse serum plus 1% fetal bovine serum). Protein expression and Cdk5 kinase activity were measured by Western blotting and in vitro kinase assay. Actin represents the internal control for protein expression. The data indicated the quantitative results of hCG-dependent effects on protein expression and kinase activity (n = 3). Control value was for 1. * and **, P < 0.05 and P < 0.01 vs. control group.

 
Cdk5 is important to testosterone production of mouse Leydig cells
Our results indicated that inhibitions of Cdk5/p35 kinase activity by inhibitors [butyrolactone-I (BL); roscovitine (RV)] and siRNA both decreased testosterone production of TM3 cells with or without hCG treatments (Fig. 3AGo). In addition, similar results were attained through treating mouse primary Leydig cells with Cdk5 inhibitors (Fig. 3BGo). Therefore, we suggest that Cdk5 might play an important role in testosterone production in mouse Leydig cells.


Figure 3
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  		FIG. 3. Testosterone production of mouse Leydig cells is Cdk5 dependent. A, Cdk5 inhibitors (BL and RV) and siRNA-cdk5 (50 pmol siRNA per 105 cells) were used to inhibit Cdk5 activity and protein expression in TM3 cells with or without hCG treatment (0.1 IU/ml). siRNA-NC, Nonspecific control of siRNA. siRNA was transfected into cells 3 d before drug treatment. B, Cdk5 inhibitors (BL and RV) were used to inhibit Cdk5 activity in mouse primary Leydig cells with or without hCG treatment (0.1 IU/ml). Testosterone concentrations in culture medium were measured by ELISA after 24 h treatment (see Materials and Methods). **, P < 0.01 vs. vehicle group; ++, P < 0.01 vs. hCG group; #, P < 0.05 vs. the group of hCG with siRNA-NC.

 
cAMP is involved in Cdk5/p35 protein expression in mouse Leydig cells
To understand the involvement of cAMP in Cdk5/p35 protein expression, the agonist [8-bromoadenosine-cAMP (8-Br-cAMP; B5386; Sigma)] and antagonist (Rp-8-Br-cAMP, B2432; Sigma) of cAMP were used. The results indicate that treatment with cAMP agonist significantly increased protein expression of Cdk5/p35 and Cdk5 activity in TM3 cells (Fig. 4AGo, lanes 1–3). However, the cAMP antagonist prevented hCG-induced increases in Cdk5/p35 protein expression and kinase activity in a dose-dependent manner (Fig. 4AGo, lanes 4–6). Interestingly, levels of StAR protein (both 30 and 37 kDa), a crucial cAMP-dependent element in steroidogenesis, corresponded to Cdk5 protein expression and kinase activity after treatments with cAMP agonist or antagonist (Fig. 4AGo). These results were quantified and shown in Fig. 4BGo (n = 3). These experiments demonstrate that Cdk5 and p35 are downstream in hCG-dependent cAMP signaling pathways. In addition, the tight correlation between Cdk5 and StAR protein implies a possible regulation mechanism in Leydig cells and need the further investigation.


Figure 4
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  		FIG. 4. cAMP is involved in the expression of Cdk5/p35 in TM3 cells. 8-Br-cAMP (0–200 µM, agonist of cAMP) was treated in culture medium for 24 h. hCG (0.1 IU/ml) was treated with Rp-8-Br-cAMP (0–200 µM, antagonist of cAMP) for 24 h. A, Protein expression and Cdk5 kinase activity were measured by Western blotting and in vitro kinase assay. Actin represents the internal control for protein expression. B, The data indicated the quantitative results of cAMP-dependent effects on protein expression and kinase activity (n = 3). Control value was for 1. * and **, P < 0.05 and P < 0.01 vs. control group; ++, P < 0.01 vs. hCG group.

 
Cdk5 increases StAR protein accumulation in mouse Leydig cells
Cdk5 knockdown (by siRNA), overexpression, and kinase inhibition (by inhibitor) were used to manipulate Cdk5 actions and the effects on StAR protein levels were determined. Regardless of whether TM3 cells were treated with hCG, StAR protein levels significantly decreased after administration of siRNA-cdk5, whereas Cdk5 protein and activity were substantially affected (Fig. 5AGo). Cdk5 inhibitor (RV, 10 µM) was also able to reduce hCG-stimulated StAR protein levels without influencing Cdk5/p35 protein levels (Fig. 5BGo). However, Cdk5 overexpression increased both Cdk5 kinase activity and StAR protein levels, with and without hCG treatments (Fig. 5CGo). Interestingly, we found that p35 protein levels were compensatively modified when Cdk5 expression was manipulated (Fig. 5CGo), which implies that homeostasis of Cdk5 kinase activity in Leydig cells is important. Because several lines of evidence indicate that StAR protein expression in Leydig cells is extremely important (20) and because Cdk5 is responsible for the stability of several proteins (26), we determined whether the Cdk5-related regulation of StAR protein is due to protein accumulation. Lactacystin (LC; proteasome inhibitor, L6785, 2 µM; Sigma) and cycloheximide (CHX; protein synthesis inhibitor, C7698, 10 µg/ml; Sigma) were used for the experiments studying accumulation of StAR protein. The data indicated that, with hCG treatments, LC treatment for 24 h could prevent Cdk5 inhibitor-reduced levels of StAR protein (Fig. 5DGo, lanes 1–4). In contrast, after 24 h treatment with Cdk5 inhibitor followed by CHX treatment for 4 additional hours, degradation of StAR protein was more significant due to Cdk5 inhibitor treatment (Fig. 5DGo, lanes 5–6). Additionally, data indicated that p35 protein in TM3 cells has a short half-life compared with Cdk5 or StAR protein after treatment with CHX (Fig. 5DGo, lanes 5–6). Together, these results suggest that Cdk5 maintains the levels of StAR protein by regulating accumulation of the protein.


Figure 5
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  		FIG. 5. StAR protein levels are dependent on Cdk5 in TM3 cells. TM3 cells were treated with or without hCG (0.1 IU/ml), siRNA-cdk5 (50 pmol siRNA per 105 cells, transfected 3 d before drug treatments), Cdk5 inhibitor (RV, 10 µM), Cdk5 overexpression (2 µg DNA per 105 cells, transfected 3 d before drug treatments), LC (2 µM), and CHX (10 µg/ml). All treatments except CHX were applied in culture medium for 24 h. A, Inhibition of Cdk5 protein expression and kinase activity by siRNA caused decreases in StAR protein levels in samples with or without hCG stimulation. B, Inhibition of Cdk5 activity by RV diminished StAR protein levels induced by hCG treatment. C, Cdk5 overexpression increased Cdk5 activity and StAR protein levels but decreased p35 protein levels in samples with or without hCG stimulation. D, LC and CHX were used to investigate the accumulation of StAR protein after treatment with RV. hCG was treated in culture medium with or without RV or LC for 24 h. CHX was applied for additional 4 h after the 24-h treatments with RV or hCG. Protein expression and Cdk5 kinase activity were measured by Western blotting and in vitro kinase assay. Actin represents the internal control for protein expression. The quantitative results of protein expression and kinase activity are shown in A–C (n = 3). Control value was for 1. * and **, P < 0.05 and P < 0.01 vs. control group; + and ++, P < 0.05 and P < 0.01 vs. hCG group.

 
Relationship between Cdk5/p35 and StAR in mouse Leydig cells
After establishing the link between Cdk5 kinase and StAR protein accumulation, our next goal was to determine whether a biochemical interaction and phosphorylation between Cdk5/p35 and StAR exist in TM3 cells. Immunoprecipitations were performed to identify interactions between these three proteins. The protein interactions of Cdk5/p35 and Cdk5/StAR were identified by coimmunoprecipitation (Fig. 6Go, A–C). However, StAR and p35 could not be immunoprecipitated with each other (Fig. 6Go, B and C). In addition, treatments with Cdk5 inhibitor decreased interactions among these three proteins, indicating that Cdk5 activity is important to these interactions (Figs. 6Go, A–C). Importantly, Cdk5 activity-dependent serine phosphorylation of StAR was observed after StAR immunoprecipitation (Fig. 6CGo). This fact might explain why Cdk5 enables to increase the accumulation of StAR protein, possibly by enhancing the phosphorylation-dependent protein stability of StAR. Figure 6DGo illustrates the subcellular distributions of Cdk5/p35 and StAR protein in TM3 cells performed by immunocytochemistry. Corresponding with the biochemical analyses, the results provide the possibility that those three proteins inside TM3 cells, particularly in the cytoplasm, interact with each other.


Figure 6
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  		FIG. 6. Cdk5 interacts with StAR protein and regulates its phosphorylation in TM3 cells. TM3 cells were treated with hCG (0.1 IU/ml) or RV (10 µM) for 24 h. A, Coimmunoprecipitations of p35 and StAR with Cdk5 were identified. IP, Immunoprecipitation. B, Coimmunoprecipitations of StAR and Cdk5 with p35 were identified. C, Coimmunoprecipitations of Cdk5 and p35 with StAR were identified. Serine-phosphorylation signals (30 and 32 kDa) of StAR were detected using anti-phosphoserine antibody. D, Immunocytochemistry was performed to show intracellular distributions of p35, Cdk5, and StAR proteins in TM3 cells. Merging different color signals demonstrated the overlaps of distribution of indicated proteins.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cdk5 and p35 are novel elements in reproductive endocrinology. Although previous reports demonstrated that Cdk5/p35 are present in Leydig cells (6, 7), there was no further evidence suggesting the biological functions of this protein complex in reproduction. In this study, we not only showed that Cdk5/p35 can regulate testosterone production in mouse Leydig cells but also demonstrated a possible mechanism in which the accumulation of StAR protein was involved in this regulation. These results imply that Cdk5 and p35 may be important factors in LH-regulated function of Leydig cells.

Since Cdk5 and its activator were identified, researchers have focused on their functions in CNS. However, several recent lines of evidence indicate that Cdk5 plays other important and interesting roles, including in cancer biology and endocrinology (4, 6, 27, 28). For instance, Cdk5 was identified as a regulator of insulin production, which was the first evidence that Cdk5 was involved in hormone production (28). Currently our focus was to research not only Cdk5 in the CNS (5) but also the roles of Cdk5 outside neuronal function in processes such as androgen production and androgen-related diseases such as prostate cancer (9). We expect that these findings will serve the development of potential treatments to androgen-related diseases.

The TM3 cell line and primary Leydig cells are considered common and useful tools for research in male reproduction. In addition, previous reports indicate that Cdk5 and p35 proteins are abundantly expressed in TM3 cells (6, 7). Therefore, these in vitro tools were appropriate tools with which we investigated the biological function of Cdk5/p35 in steroidogenesis. The regulated-expression of Cdk5/p35 was first confirmed by treating cells with hCG, a trophic hormone of Leydig cells that activates the LH receptor. Cdk5/p35 protein expression and kinase activity were found to be regulated by hCG (Figs. 1Go and 2Go), which is consistent with previous reports (6). Importantly, Cdk5-dependent androgen production was identified in low serum conditions (1% horse serum + 1% fetal bovine serum). However, the Cdk5 effects on androgen production were not significant under normal culture conditions (data not shown). Therefore, the optimal conditions (hCG treatment: 0.1 IU/ml, 24 h in low serum condition) for hCG-induced stimulation of Cdk5/p35 expression and androgen production were used throughout our experiments to investigate Cdk5 effects on androgen production. In addition, Cdk5 inhibitors were used in primary Leydig cells (Fig. 3BGo), StAR protein stability (Fig. 5Go), and the issue of protein interaction (Fig. 6Go). To verify the specificity of Cdk5 inhibitor to other member of Cdk proteins, Cdk5 inhibition test was performed. The data indicated that RV inhibited Cdk5 activity at the concentrations of 5 and 10 µM for 24 h treatment in TM3 cells but not Cdc2 (supplemental Fig. 2). Our results demonstrate that Cdk5/p35 is tightly regulated and probably involved in testosterone production: the main function of Leydig cells. Indeed, manipulation of Cdk5 protein expression and activity affected testosterone production by Leydig cells (Fig. 3Go). Although several lines of evidence indicate that peptide exocytosis is regulated by Cdk5 in endocrine cells (29, 30), we found that intracellular testosterone production of TM3 cells corresponded to the amount released into the culture medium (supplemental Fig. 3). This suggests that detection of testosterone secretion in culture medium can be used as an indicator for testosterone production in Leydig cells. These facts contribute to the first solid evidence indicating the involvement of Cdk5 in androgen biosynthesis.

Because Cdk5 involves hCG-dependent production of testosterone in mouse Leydig cells, cAMP, a crucial second messenger of the LH/hCG receptor, probably controls steroidogenesis through Cdk5 regulation. Indeed, we found that Cdk5/p35 expression was affected by cAMP and its antagonist, suggesting that cAMP is involved in signaling between hCG stimulation (i.e. LH receptor activation) and Cdk5/p35 expression (Fig. 4Go). However, protein levels of StAR were observed to correspond with the regulated levels of Cdk5/p35 (Fig. 4Go). This implies that StAR protein is directly involved in Cdk5-dependent regulation of testosterone production. Therefore, the connection between StAR and Cdk5 is an important issue to further investigate. Because no reports indicate a relationship between StAR expression and any Cdks in Leydig cells, our strategy was to alter the activity and expression of Cdk5 and subsequently measure the effects on StAR protein expression. Interestingly, StAR protein levels were correlated to not only Cdk5 protein levels but also its activity (Fig. 5Go, A–C). Testosterone production in culture medium was also found to be regulated by changes of Cdk5 and StAR (supplemental Fig. 4). Figure 5DGo indicates that the Cdk5-dependent increases in the levels of StAR protein were due to accumulation of stable proteins but not transcription, as detected by semiquantitative RT-PCR (supplemental Fig. 5). Taken together, increasing the accumulation of the StAR protein is probably a mechanism by which Cdk5 regulates steroidogenesis in mouse Leydig cells. On the other hand, our previous results indicated the involvement of Cdk5/p35 in cell proliferation and apoptosis (4, 5, 9). However, the data showed that Cdk5 inhibition (by inhibitor or siRNA) did not affect proliferation of TM3 cells after 24 h treatment (supplemental Fig. 6). Therefore, our findings that indicate the role of Cdk5/p35 in testosterone production in 24 h can exclude the influence of cell proliferation.

Because Cdk5 is able to regulate protein stability through phosphorylation (26) and because phosphorylation of StAR is important to steroidogenesis (31, 32), the novel relationship (protein interaction and phosphorylation) between Cdk5 and StAR is an important issue to be studied further. Our results suggest a possible mechanism by which Cdk5 enhances accumulation of StAR protein through Cdk5-dependent phosphorylation (StAR). However, we found that coimmunoprecipitation of StAR and p35 did not occur. In addition, p35 knockdown decreased both StAR phosphorylation and Cdk5/StAR interaction (supplemental Fig. 7). A possible explanation is that StAR and p35 compete with each other for similar (or partially overlapping) interacting domains of Cdk5. This speculation should be tested in future studies.

In conclusion, our findings are the first evidence that Cdk5/p35 plays a role in androgenic steroidogenesis. We suggest that Cdk5 is probably important in routine control of androgen production through posttranslational regulation of StAR. We hope these findings will be helpful in not only reproductive endocrinology but also treating androgen-related diseases through manipulation of the upstream androgen source.


   Acknowledgments
 
The authors thank Dr. Shih-Lan Hsu (Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan) and Professor Paulus S. Wang (Department of Physiology, National Yang Ming University, Taipei, Taiwan) for full support. The authors also thank Dr. Walter L. Miller, University of California, San Francisco, for kindly providing antibody against StAR protein.


   Footnotes
 
This work was supported by National Science Council Grant NSC94-2320-B-005-006 and the Ministry of Education (under the Aiming for the Top University plan), Taiwan, Republic of China.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 28, 2008

Abbreviations: BL, Butyrolactone-I; 8-Br-cAMP, 8-bromoadenosine-cAMP; Cdk, cyclin-dependent kinase; CHX, cycloheximide; CNS, central nervous system; hCG, human chorionic gonadotrophin; LC, lactacystin; RV, roscovitine; siRNA, small interfering RNA; StAR, steroidogenic acute regulatory.

Received April 8, 2008.

Accepted for publication August 20, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. R. Manna, M. T. Dyson, and D. M. Stocco
Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives
Mol. Hum. Reprod., June 1, 2009; 15(6): 321 - 333.
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