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Biology of Reproduction 67, 789-794 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Src Tyrosine Kinase-Induced Loss of Luteinizing Hormone Responsiveness Is via a Ras-Dependent, Phosphatidylinositol-3-Kinase Independent Pathway1

Christopher C. Taylor2,a

a Department of Cell Biology, Georgetown University School of Medicine, Washington, District of Columbia 20007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropins stimulate gonadal cell steroid secretion primarily through activation of a cAMP-protein kinase A signal transduction pathway. Various growth factors have been shown to inhibit gonadotropin-stimulated steroidogenesis, however, the intracellular signaling cascades involved in growth factor inhibition have not been characterized. The present study investigated whether Src tyrosine kinase, a nonreceptor tyrosine kinase activated in response to growth factor stimulation and previously shown to inhibit LH-stimulated progesterone secretion, acts via activation of Ras stimulated pathways, phosphatidylinositol-3-kinase (PI3-kinase) stimulated pathways, or both in MA10 Leydig cells. Direct activation of Src in MA10 cells that express a temperature sensitive Src was associated with an increase in GTP-bound Ras, indicating increased Ras activity in response to Src activation. Direct activation of Ras by way of expression of a constitutively active Ras (Ras+) was associated with a decrease in LH responsiveness. Coexpression of a dominant negative Src, which by itself increases LH responsiveness in MA10 cells, had no effect on Ras+ inhibition on LH responsiveness, further demonstrating that Src is upstream of Ras. In addition, MA10Ras+ cells were relatively unresponsive to cholera toxin or 8-bromo cAMP, indicating the effects of Ras are independent of cAMP generation. Wortmannin, a PI3-kinase inhibitor, did not restore LH responsiveness to cells expressing activated Src or constitutively active Ras. These results demonstrate that Src activates a Ras pathway in MA10 Leydig cells, and that activation of Ras is associated with a loss of LH responsiveness that is independent of PI3-kinase.

growth factors, kinases, Leydig cells, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropic hormones stimulate gonadal steroidogenic cells to synthesize and secrete sex steroid hormones. Both LH and FSH bind specifically to their respective seven transmembrane-spanning G-protein coupled receptors(LH-R and FSH-R). Both the LH-R and FSH-R are primarily coupled to Gs{alpha} which, upon activation, stimulates adenylate cyclase, leading to increased intracellular cAMP and activation of cAMP-dependent protein kinase A, with the end result of increased steroidogenesis and steroid secretion. Many studies have demonstrated that growth factors such as epidermal growth factor [1, 2], platelet-derived growth factor [3, 4], and the fibroblast growth factors [5] modulate gonadotropin-stimulated steroid secretion by gonadal steroidogenic cells, however, the mechanisms by which growth signals modulate steroidogenesis have not been well delineated.

Previous reports demonstrated that herbimycin A, a specific inhibitor of the Src family of nonreceptor tyrosine kinases, potentiated LH-stimulated progesterone secretion by rat thecal-interstitial cells [6]. Herbimycin was found to indirectly inhibit cAMP-specific phosphodiesterase activity, thus increasing LH-stimulated cAMP accumulation [6, 7]. Furthermore, it was found that expression of a dominant negative Src tyrosine kinase in MA10 Leydig cells was associated with an increase in LH responsiveness and a decrease in phosphodiesterase activity, whereas expression of a temperature sensitive Src was associated with a decrease in LH responsiveness at the Src active temperature [8]. Thus it appears that Src tyrosine kinase activity modulates LH-stimulated progesterone secretion, at least in part by modulation of phosphodiesterase activity. However, it is also likely that Src is involved in activating growth factor signaling cascades that may also affect the cAMP-protein kinase A pathway activated by LH.

In many cell types, Src is known to activate phosphatidylinositol-3-kinase (PI3-kinase), a ubiquitous kinase that phosphorylates phosphoinositides at the D-3 position of the inositol ring [9]. Phosphatidylinositol-3,4,5-triphosphate, the most abundant metabolite of PI3-kinase activity, is a potent stimulator of PKC{zeta} [10], a ubiquitous [11] Ca2+-independent, diacylglycerol-insensitive protein kinase C isoform [12]. However, Src is also believed to act through Ras [13], and there is evidence to suggest that activation of PI3-kinase is via a Ras-dependent mechanism [14]. Conversely, activation of Ras in some systems appears to be downstream of PI3-kinase [15], with Ras directly activating PKC{zeta} [16].

The present studies were undertaken in order to determine whether Src might be acting via a Ras pathway, a PI3-kinase pathway, or both. Cell lines expressing either a dominant negative Src or a temperature sensitive Src tyrosine kinase that have been previously described [8] were utilized. In addition, new lines expressing a constitutively active Ras with or without a dominant negative Src kinase were created. The results indicate that Src activates Ras and that Ras activation is associated with lower progesterone secretion in response to LH stimulation. The loss of LH responsiveness is independent of PI3-kinase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

All liquid media, G418 sulfate, and lipofectamine were purchased from Life Technologies (Grand Island, NY). A panspecific Ras antibody (clone Y13-259) and protein AG+ agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An Src-specific antibody (clone 327) was purchased from Oncogene Science (Cambridge, MA). Antibodies specific for phospho-Akt and total Akt were purchased from Cell Signaling Technology (Beverly, MA). A Ras activation assay kit was from Upstate Biotechnology (Lake Placid, NY). Ovine LH (LH S-25; 2.3 U/mg) was obtained from the National Hormone and Pituitary Program (Bethesda, MD). Wortmannin was obtained from Calbiochem (La Jolla, CA). [32P]Orthophosphate (500 mCi/ml) was obtained from ICN (Costa Mesa, CA). Enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham Life Science (Arlington Heights, IL). All other reagents were purchased from Sigma (St. Louis, MO).

Constructs

A modified pSG5 vector containing a sequence encoding a dominant negative Src kinase (Srck-) under the control of the simian SV40 promoter was generously provided by Sara Courtneidge (The Van Andel Research Institute, Grand Rapids, MI). The kinase inactivating mutation was Lys 295 -> Met [17]. A pLNCX plasmid containing a G418 resistance gene and a sequence encoding a temperature sensitive Src kinase (tsUP) under the control of the Maloney murine leukemia virus promoter was kindly provided by Joan Brugge (Harvard University, Cambridge, MA). A mutation of Glu 280 -> Lys results in a viral Src protein that is active at 35°C and inactive at 39.5°C [18]. A pSVras construct encoding the oncogenic valine 12 mutant Ha-ras under the control of the SV40 early promoter was provided by Arthur Gutierrez-Hartmann (University of Colorado Health Sciences Center, Denver, CO). A pSV2neo plasmid to convey G418 resistance was generously provided by Michael Soares (University of Kansas Medical Center, Kansas City, KS).

Cell Lines

MA10 Leydig tumor cells were originally provided by Mario Ascoli (University of Iowa, Iowa City, IA). The MA10pSV2neo, MA10Srck-2 and MA10SrctsUP cell lines were produced as previously described [8]. Cells were cultured in Dulbecco modified Eagle medium (DMEM)/Ham F12 1:1, 15 mM Hepes, 5% horse serum, 2.5% fetal bovine serum, and 200 µg/ml of G418 sulfate. All cell lines were routinely maintained at 37°C in a water-saturated atmosphere of 95% air and 5% CO2, except for MA10SrctsUP cells, which were maintained at 39.5°C, the temperature sensitive Src inactive temperature.

MA10Ras+ and MA10Src-Ras+ cells were produced by cotransfecting MA10 Leydig cells with pSVras and pSV2neo (MA10Ras+ cells) and pSG5srck-, pSVras and pSV2neo (MA10Src-Ras+ cells) using lipofectamine according to the protocol of the manufacturer (Life Technologies). Briefly, MA10 cells were plated at 2–4 x 105 cells/ml in serum containing medium (without G418) as described above. The following day, cells were washed two times with serum-free, antibiotic-free media. Media were removed and replaced with 1 ml of serum-free, antibiotic-free media containing lipofectamine and DNA complexes (1.0 µg of pSVras and 0.1 µg of pSV2neo, or 1.0 µg of pSG5srck-, 1.0 µg of pSVras, and 0.15 µg pSV2neo) and allowed to incubate for 5 h. After 5 h, 1 ml of medium plus serum was added to each well. Following 72 h of incubation, media were changed to fresh serum containing media plus 200 µg/ml of G418 sulfate. Over the next several weeks, G418-resistant clones were selected and characterized for Ras and Src expression. All clones were maintained in media containing G418 sulfate.

To determine progesterone secretion, cell lines were plated in tissue culture plates (20 000 cells/cm2) in serum containing DMEM/Ham F12 media. Cells were allowed to attach and grow for approximately 32 h, and then media were changed to fresh serum-free, antibiotic-free media. Sixteen hours later, cells were rinsed and fresh serum-free, antibiotic-free media were added. Cells were then challenged with ovine LH (oLH NIH-25; 50 ng/ml), 8 bromo-cAMP (1 mM), cholera toxin (500 ng/ml), or vehicle (1% PBS in media) as specified in Results. When the kinase inhibitor wortmannin was used, cells were preincubated with wortmannin (10–100 nM) or vehicle (0.5% DMSO) for 16 h before stimulation with LH. Wortmannin or vehicle remained in the media during the LH stimulation period. At the end of the LH stimulation period, media were collected for progesterone determination by radioimmunoassay as previously described [19]. Intraassay and interassay coefficients of variation were 6% and 11%, respectively. The limit of detection was 0.25 ng/ml.

Determination of Ras Activity

The ratio GDP-bound:GTP-bound Ras was used to determine cellular Ras activity [20]. Cells were plated in T75 flasks and allowed to grow until they were approximately 75% confluent. Cells were then serum starved for 16 h, labeled with [32P]orthophosphate (0.5 mCi/ml for 4 h) in phosphate-free media, and subsequently lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). Cellular lysates were cleared of insoluble material by centrifugation (14 000 x g for 20 min). Anti-Ras antibody (1 µg) was added to the total soluble lysate and incubated at 4°C for 2 h with constant agitation. Protein AG + agarose (20 µl) was then added, and the lysates were further incubated overnight at 4°C with constant agitation. Immunoprecipitates were collected by centrifugation (14 000 x g for 2 min at 4°C), washed extensively, and resuspended in 20 µl of 20 mM Tris-HCl pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP. Ras-bound guanine nucleotides were eluted by heating to 65°C for 5 min and centrifuging at 14 000 x g for 5 min. The eluted guanine nucleotides were then separated by spotting 5 µl of supernatant on polyethyleneimine cellulose thin layer chromatography (TLC) plates and developing with 0.75 M KH2PO4 (pH 3.4). Following autoradiography of the TLC plates, GDP and GTP spots were isolated from the plates and quantified by liquid scintillation counting. The percentage of GTP-bound Ras was calculated by GTPcpm/[(GDPcpm x 1.5) + GTPcpm].

Nonradioactive Ras activation assays utilizing a Raf-1-GST-agarose conjugate fusion protein containing the Ras binding domain of Raf-1, and which binds only activated GTP-bound Ras, were also performed. At the end of the treatment period, cells were lysed in Ras lysis buffer (25 mM Hepes pH 7.5, 150 mM NaCl, 1% NP-40, 100 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM PMSF, and 1 mM sodium orthovanadate). Cellular protein (500 µg) was mixed with 5 µl of a Raf-1-GST-agarose conjugate fusion protein containing the Ras binding domain of Raf-1, and which binds only activated GTP-bound Ras. The mixture was allowed to incubate overnight at 4°C with gentle agitation. Agarose beads were collected and washed three times with lysis buffer. Agarose beads were then resuspended in 2x Laemmli sample buffer, heated to 95°C for 5 min, and centrifuged at 14 000 x g. The supernatant was then subjected to SDS-PAGE (12% gel) and immunoblot analysis as described above with an antibody specific for Ras.

Statistical Analysis

Unless otherwise specified, the results are presented as means ± standard errors (SEM) from representative experiments. Three replicates per treatment were used for each experiment. For statistical analysis, means across treatments were subjected to an analysis of variance with differences between individual means compared by a Fisher protected least significant differences test (Statview 512+, Agoura Hills, CA). Experiments were repeated a minimum of three times in order to determine relative consistency of results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of Src Leads to Increased Ras Activity

In order to determine whether Src activates Ras, MA10pSV2neo cells (control cells) and MA10Src-tsUP cells were cultured at 39.5°C (the temperature sensitive Src inactive temperature), 39.5°C then shifted to 35°C (the Src active temperature) for 1 h, or they were maintained at 35°C. The percentage of GTP-bound Ras in MA10pSV2neo cells was relatively low (approximately 5%), indicating a low level of Ras activity, and was unaffected by the culture temperature (Fig. 1). MA10Src-tsUP cells cultured at 39.5°C also had relatively low levels of GTP-bound Ras, however, shifting MA10Src-tsUP cells to 35°C for 1 h was associated with a doubling of GTP-bound Ras, indicating Ras activation. Maintaining MA10Src-tsUP cells at 35°C was associated with an even greater increase in the proportion of GTP-bound Ras (approximately 20%). As confirmation, a second type of Ras activation assay was employed. GTP-bound Ras was precipitated with a fusion protein containing the Ras binding domain of Raf-1, which binds only activated GTP-bound Ras. Again, temperature had no effect on the proportion of precipitated GTP-bound Ras in MA10pSV2neo cells. In contrast, precipitated GTP-bound Ras increased in MA10Src-tsUP cells that were shifted to 35°C for 1 h or maintained at 35°C compared with MA10Src-tsUP cells maintained at 39.5°C. These results demonstrate that Src can activate Ras in MA10 cells.



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  		FIG. 1. Activation of Src is associated with activation of Ras. MA10 control cells (MA10pSV2neo) and MA10 cells transfected with a temperature sensitive Src (MA10Src-tsUP) were cultured at the temperature sensitive Src inactive temperature (39.5°C), 39.5°C then shifted to the Src active temperature (35°C) for 1 h, or maintained at the Src activation temperature of 35°C. The ratio of GDP-bound to GTP-bound Ras was used to determine cellular Ras activity as outlined in Materials and Methods. A representative autoradiography of separated GTP and GDP (a), a representative immunoblot of GTP-bound Ras (b), and the fold increase (standard error of the mean) in GTP-bound Ras with the change in temperature across three separate in vitro labeling experiments (c) are shown. GTP-bound Ras at 39.5°C was set to 1. Bars with different letters are significantly different (P < 0.05)

Ras Activation Inhibits LH-Stimulated Progesterone Secretion

It was previously demonstrated that inhibition of Src in thecal-interstitial cells and MA10 Leydig cells was associated with a higher responsiveness to LH stimulation with regard to cAMP and progesterone secretion, whereas activation of Src was associated with a loss of responsiveness [8]. In order to determine whether Ras is involved in this loss of responsiveness, MA10 Leydig cell lines expressing a constitutively active Ras (MA10Ras+) and cells coexpressing a dominant negative Src and a constitutively active Ras (MA10Src-Ras+) were created. Immunoprecipitation and immunoblot analysis with an antibody specific for Src demonstrates high expression of Src protein in MA10Src-Ras+ compared to MA10pSV2Neo control cells and MA10Ras+ cells, indicating expression of the Src dominant negative (Fig. 2a). Two clonal lines of each MA10Ras+ and MA10Src-Ras+ cells show greatly elevated proportions of GTP-bound Ras, indicating expression of the constitutively active Ras (Fig. 2, b and c; results from only one cell line shown). Coexpression of the dominant negative Src had no effect on the proportion of GTP-bound Ras in cells expressing the constitutively active Ras (MA10Src-Ras+ cells; Fig. 2).



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  		FIG. 2. Expression of constitutively active Ras and an Src dominant negative in MA10 Leydig cells. Cells were transfected with either a constitutively active Ras (Ras+2) or dominant negative Src and a constitutively active Ras construct (Src-/Ras+1). Cells were lysed and analyzed for Src expression by immunoblot analysis (a) and the ratio of GDP-bound to GTP-bound Ras (b, c). The bar graph represents means ± SEM of the percentage of GTP-bound Ras across three separate experiments. Bars with different letters are significantly different (P < 0.05)

As previously reported, MA10Srck-2 cells have an even greater response to LH stimulation than the MA10pSV2neo control cells (Fig. 3). Conversely, MA10Ras+ cells show a large attenuation of LH-stimulated progesterone secretion, thus demonstrating that direct activation of Ras is associated with a loss of LH responsiveness. Furthermore, coexpression of the Src dominant negative in MA10Src-Ras+ cells did not rescue or reverse the effects of expression of the constitutively active Ras, providing further support for our observation that Src is upstream of Ras.



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  		FIG. 3. LH-stimulated progesterone secretion (standard errors of the mean) by MA10 Leydig clonal cell lines expressing a dominant negative Src kinase (SrcK-2), a constitutively active Ras (Ras+1, Ras+2), or a dominant negative Src and a constitutively active Ras (Src-Ras+1, Src-Ras+2). Cells were serum starved for 16 h and then treated with 50 ng/ml of ovine LH for 4 h. Media were collected and analyzed for progesterone by specific radioimmunoassay. Bars with different letters are significantly different (P < 0.05)

In order to determine whether the effects of Ras activation were on cAMP generation, MA10pSV2neo and MA10Ras+ cells were stimulated with either cholera toxin or 8-bromo cAMP. MA10Ras+ cells again had an attenuated response to both cholera toxin and 8-bromo cAMP compared to the MA10pSV2neo control cells (Fig. 4), indicating the effects of Ras activation are distal to or independent of cAMP generation.



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  		FIG. 4. Ras activation inhibits MA10 steroid secretion distal to cAMP generation. Control MA10 Leydig cells (Neo) and MA10 cell lines expressing a constitutively active Ras (Ras+1 and Ras+2) were serum starved for 16 h and then stimulated with either ovine LH, cholera toxin, or 8-bromo cAMP for 2 h. Media were then collected and analyzed for progesterone by specific radioimmunoassay. Data represent means ± SEM of three replicates within a representative experiment. Experiments were repeated a minimum of three times with qualitatively similar results

Wortmannin Does Not Rescue MA10 Cells from Src or Ras Activation

Src activation is often associated with activation of PI3-kinase, which may or may not be via Ras. Our various cell lines were treated with the PI3-kinase inhibitor wortmannin (16 h of pretreatment) in order to determine whether PI3-kinase was involved in the Src/Ras desensitization of MA10 Leydig cells to LH stimulation. As expected, culture of MA10Src-tsUP cells at the Src active temperature inhibited LH-stimulated progesterone secretion compared to MA10pSV2neo control cells cultured at the same temperature (Fig. 5). MA10Src-tsUP cells pretreated with wortmannin (100 nM) remained relatively unresponsive to LH stimulation, indicating that PI3-kinase is not involved in the Src induced loss of LH responsiveness. Similarly, wortmannin (10 to 100 nM) was unable to reverse the effects of Ras activation in MA10Ras+ cells (Fig. 6). Importantly, wortmannin (50–100 nM) completely inhibited Akt phosphorylation, demonstrating inhibition of PI3-kinase activity (Fig. 6). Results were further confirmed with a second PI3-kinase inhibitor, LY294002 (data not shown).



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  		FIG. 5. Wortmannin does not restore progesterone secretion in MA10 Leydig cells with activated Src. Control MA10 Leydig cells (Neo) and an MA10 cell line expressing a temperature sensitive Src (tsUP) were cultured at either the Src inactive temperature (39.5°C) or the Src activation temperature (35°C). Cells were serum starved and pretreated with the PI3-kinase inhibitor wortmannin for 16 h, and then stimulated with ovine LH (50 ng/ml) for 2 h. Media were collected and analyzed for progesterone by specific radioimmunoassay. Data represent means ± SEM of three replicates within a representative experiment. Experiments were repeated a minimum of three times with qualitatively similar results. Bars with different letters are significantly different (P < 0.05)



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  		FIG. 6. Wortmannin does not restore progesterone secretion in MA10 Leydig cells with activated Ras. Control MA10 cells (Neo) and MA10 cell lines expressing a constitutively active Ras (Ras+1 and Ras+2) were cultured at 37°C. Cells were serum starved and pretreated with the PI3-kinase inhibitor wortmannin for 16 h, and then stimulated with ovine LH (50 ng/ml) for 2 h. Cells were collected and lysed, and lysates were analyzed by immunoblot analysis for phosphorylated Akt and total Akt expression (a). In separate experiments, media were collected and analyzed for progesterone by specific radioimmunoassay (b). Data represent means ± SEM of three replicates within a representative experiment (b). Experiments were repeated a minimum of three times with qualitatively similar results


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Numerous reports have demonstrated that treatment of gonadal steroidogenic cells with growth factors such as epidermal growth factor (EGF), platelet-derived growth factors (PDGFs), and fibroblast growth factor results in an attenuation of gonadotropin-stimulated steroid secretion [1–5]. The mechanisms by which growth factor stimulation modulates steroidogenesis have yet to be fully elucidated. Many receptor tyrosine kinases, such as the EGF receptor, signal at least partially through the activation of the small G protein, Ras. Results from the present study indicate that Src tyrosine kinase, an intracellular kinase activated by numerous growth factor receptors, can activate Ras in MA10 Leydig cells. In addition, the present study demonstrates for the first time that direct activation of Ras by way of expression of a constitutively activated Ras is associated with a loss of responsiveness to LH stimulation.

These findings confirm and extend our previous reports that activation of Src inhibits LH-stimulated progesterone secretion [8], whereas inhibition of Src potentiates LH stimulation [6, 8]. They also suggest that growth factors that activate Ras can inhibit LH-stimulated progesterone secretion independent of cAMP generation. Treatment of MA10Ras+ and MA10Src-Ras+ cells were relatively unresponsive to either cholera toxin or 8-bromo cyclic AMP. This is somewhat at odds with a previous report that EGF-induced desensitization of MA10 cells was completely accounted for by a decrease in LH receptor number and that EGF pretreated cells were fully responsive to cholera toxin and 8-bromo cAMP [2]. However, that report also demonstrated that the EGF receptor was rapidly internalized, perhaps terminating the EGF signal, whereas LH receptor down-regulation and replenishment lagged far behind. The present work utilized a constitutively active Ras, resulting in a constitutively activated growth signaling pathway. It is interesting that MA10Ras+ cells could be further desensitized with LH pretreatment, but EGF pretreatment did not result in further desensitization (unpublished observations). A recent report demonstrates that inhibition of mitogen-activated protein kinase kinase (MEK), a downstream effector of Ras, is associated with increased gonadotropin-stimulated steroidogenesis. This appears to be the result of increased intracellular steroidogenic acute regulator protein (StAR) content [21].

As demonstrated in the present study, direct activation of Src can lead to activation of Ras. However, whether Src is required for growth factor activation of Ras is uncertain. Src is believed to phosphorylate secondary tyrosine sites within the cytoplasmic tail of growth factor receptors such as PDGF-Rß [22]. A previous study utilizing primary cultures of porcine thecal cells reported that herbimycin A, an Src selective inhibitor, blocked PDGF receptor activation [4] and thus Ras activation. However, it was not clear whether the effects of herbimycin A were direct effects on the receptor or through inhibition of Src, blocking secondary PDGF receptor tyrosine phosphorylation.

A common consequence of Src-Ras activation is the activation of PI3-kinase. Wortmannin, a PI3-kinase inhibitor, did not revert MA10 cells in which Src or Ras had been activated, to a more LH responsive phenotype, suggesting that PI3-kinase activation is not important in the Src-Ras induced loss of LH responsiveness. Akt phosphorylation is a measure of PI3-kinase activity. Wortmannin treatment inhibited Akt phosphorylation in a dose-dependent manner, demonstrating PI3-kinase inhibition by wortmannin. These findings are in accordance with previous reports that wortmannin has no effect on steroidogenesis in porcine thecal cells treated with PDGF-BB and LH [4].

How activation of a Src-Ras pathway is inhibiting steroidogenesis is not clear but is probably via multiple mechanisms, both before and after cAMP generation. Various studies have demonstrated that growth factor stimulation decreases LH-receptor content [1, 2] and steroidogenic enzyme expression [2, 23]. There is also evidence that activation of MEK and extracellular-regulated kinase, downstream effectors of Ras activation, are associated with a decrease in StAR protein [21]. At another level, activation of Src and Ras may be important for G-protein deactivation. Although the evidence is still limited, there is speculation that regulators of G-protein signaling are activated by Ras or Ras-associated proteins [24, 25]. In addition, Src has been shown to phosphorylate G protein-coupled receptor kinase 2 (GRK2), ultimately deactivating the G protein signal [26]. Finally, G protein-coupled receptors, including LH receptors, are internalized by an arrestin-dependent mechanism [27–29]. Src appears to be necessary for this arrestin-dynamin mediated G protein-coupled receptor desensitization [30, 31]. Thus, constitutive activation of Src may be associated with ligand-independent LH receptor desensitization.

In conclusion, activation of Src tyrosine kinase in MA10 Leydig cells is associated with Ras activation. This in turn desensitizes MA10 cells to LH stimulation with regard to steroid secretion. This desensitization is independent of PI3-kinase activation.


   FOOTNOTES
 
First decision: 16 November 2001.

1 Supported by grant HD-36013 from the National Institutes of Health. Back

2 Correspondence: Chris Taylor, Department of Cell Biology, Georgetown University School of Medicine, 3900 Reservoir Road, Washington, DC 20007. FAX: 202 687 1823; cct5{at}georgetown.edu Back

Accepted: April 2, 2002.

Received: October 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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