The Lutropin/Choriogonadotropin Receptor-Induced Phosphorylation of the Extracellular Signal-Regulated Kinases in Leydig Cells Is Mediated by a Protein Kinase A-Dependent Activation of Ras

Takashi Hirakawa and Mario Ascoli

Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242

Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2-319B BSB, 51 Newton Road, University of Iowa, Iowa City, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pathways involved in activation of the ERK1/2 cascade in Leydig cells were examined in MA-10 cells expressing the recombinant human LH receptor (hLHR) and in primary cultures of rat Leydig cell precursors. In MA-10 cells expressing the recombinant hLHR, human choriogonadotropin-induced activation of ERK1/2 is effectively inhibited by overexpression of a cAMP phosphodiesterase (a manipulation that blunts the human choriogonadotropin-induced cAMP response), by addition of H89 (a selective inhibitor of protein kinase A), or by overexpression of the heat-stable protein kinase A inhibitor, but not by overexpression of an inactive mutant of this inhibitor. Stimulation of hLHR did not activate Rap1, but activated Ras in an H89-sensitive fashion. Addition of H89 to MA-10 cells that had been cotransfected with a guanosine triphosphatase-deficient mutant of Ras almost completely inhibited the hLHR-mediated activation of ERK1/2. We also show that 8-bromo-cAMP activates Ras and ERK1/2 in MA-10 cells and in primary cultures of rat Leydig cells precursors in an H89-sensitive fashion, whereas a cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP (8CPT-2Me-cAMP) that is selective for cAMP-dependent guanine nucleotide exchange factor has no effect. Collectively, our results show that the hLHR-induced phosphorylation of ERK1/2 in Leydig cells is mediated by a protein kinase A-dependent activation of Ras.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PHENOTYPE OF 46,XY individuals harboring germ-line loss of function or gain of function mutations of the human LH receptor (hLHR) clearly shows that this receptor is important for the proliferation and differentiation of Leydig cells (reviewed in Refs.1, 2, 3). As such, 46,XY individuals harboring germ-line gain of function mutations have Leydig cell hyperplasia, whereas those harboring germ-line loss of function mutations of the hLHR have Leydig cell hypoplasia. Moreover, the finding of a somatic gain of function mutation of the hLHR in Leydig cell tumors of boys with precocious puberty (4, 5) suggests that the LHR may even be involved in the neoplastic transformation of this cell type. It is, therefore, important to understand the molecular basis of the LHR-induced activation of mitogenic signaling pathways in Leydig cells.

When expressed in heterologous cell lines of different species (such as 293, COS-7, or L cells) or in a mouse Leydig tumor cell line (MA-10), the agonist-engaged hLHR activates several families of G proteins and signaling events, such as cAMP and inositol phosphate accumulation and the phosphorylation of ERK1/2 (reviewed in Ref.3). The agonist-induced activation of hLHR also enhances its association with the nonvisual arrestins and the agonist-engaged hLHR is internalized by a nonvisual arrestin- and dynamin-dependent pathway (reviewed in Ref.3). An agonist-independent activation of all of these signaling and trafficking events can be faithfully reproduced by gain of function germ-line mutations of the hLHR found in individuals with Leydig cell hyperplasia or by a somatic mutation of the hLHR found in Leydig cell tumors (6, 7, 8). Thus, it is possible that any of these pathways could be responsible for the LHR-induced proliferation of Leydig cells.

Although much is known about the events leading to the LHR-induced activation of cAMP and inositol phosphate accumulation (reviewed in Ref.3), our knowledge about the mechanisms by which activation of LHR leads to an increase in ERK1/2 phosphorylation is more limited. For example, based on the coexpression of constructs that sequester Gß/{gamma}-subunits, it was concluded that LHR-mediated stimulation of ERK1/2 phosphorylation in a heterologous cell type (COS-7 cells) is mediated by Gß/{gamma} (9). In contrast, studies with primary cultures of porcine (10) or rat (11, 12) granulosa cells as well as human granulosa/luteal cells (13) or with immortalized rat granulosa cell lines expressing the recombinant rat LHR (14) indicate that LHR-mediated activation of the ERK1/2 cascade in ovarian cells is a cAMP/protein kinase A (PKA)-dependent process. The mechanisms by which PKA activation leads to the phosphorylation of ERK1/2 in ovarian cells were not investigated, however. Moreover, it is not known whether the LHR-induced phosphorylation of ERK1/2 in ovarian and Leydig cells occurs by the same or different mechanisms. These questions need to be addressed because the effects of cAMP and PKA on the ERK1/2 cascade are complex, and they are often cell type specific (reviewed in Ref.15). With this in mind, the studies presented here were designed to understand the pathway(s) by which the LHR activates the ERK1/2 cascade in Leydig cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Roles of Second Messengers in hLHR-Induced Phosphorylation of ERK1/2 in MA-10 Cells
MA-10 cells are a clonal strain of mouse Leydig tumor cells that retain many of the differentiated functions of their normal counterparts, including the expression of low levels (~1000 receptors/cell) of endogenous LHR (6, 16, 17). Although activation of the endogenous LHR present in MA-10 cells can mediate a 2- to 3-fold increase in the phosphorylation of ERK1/2, transient expression of the hLHR results in a 7- to 15-fold increase in ERK1/2 phosphorylation (see below) (6). Therefore, in all of the experiments described below we used MA-10 cells transiently transfected with wild-type hLHR (hLHR-wt; to attain a density of ~100,000 receptors/cell) (6) to better delineate the pathways by which the activation of this receptor leads to an increase in the phosphorylation of ERK1/2.

The addition of human choriogonadotropin (hCG) to hLHR-transfected MA-10 cells results in the activation of Gs, Gi/o, and Gq/11 (8) and subsequent activation of the cAMP and inositol phosphate/diacylglycerol signaling cascades (6). As these pathways can promote the phosphorylation of ERK1/2 (18), we initially tested their involvement in the phosphorylation of ERK1/2 in MA-10 cells by using 8-bromo-cAMP (8Br-cAMP) and phorbol 12-myristate 13-acetate (PMA) as surrogates of the second messengers generated in response to hLHR activation. Fig. 1Go shows that, when used at maximally effective concentrations, 8Br-cAMP and PMA induce MEK and ERK1/2 phosphorylation in MA-10 cells to about the same extent as that provoked by a maximally effective concentration of hCG. The involvement of MEK activation in the phosphorylation of ERK1/2 elicited by these three stimuli could be readily documented with the use of U0126, a selective MEK inhibitor (19). This compound completely inhibited the 8Br-cAMP-, PMA-, or hCG-induced increase in ERK1/2 phosphorylation (Fig. 1Go).



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Fig. 1. Second Messenger-Induced Phosphorylation of MEK and ERK1/2 in MA-10 Cells

MA-10 cells transiently transfected with the hLHR-wt (1 µg of plasmid/35-mm well) were preincubated with or without 10 µM U0126 for 30 min as indicated and then further incubated with buffer only, hCG (3 nM for 30 min), 8Br-cAMP (1 mM for 15 min), or PMA (100 nM for 15 min). Only the relevant portions of representative phospho-MEK (A) or phospho-ERK1/2 (B and C) blots are shown. Total ERK1/2 and MEK levels did not change (data not shown).

 
We have previously reported that the 50% effective concentration for the hCG-induced phosphorylation of ERK1/2 in hLHR-transfected MA-10 cells is approximately 0.4 nM (6). 8Br-cAMP and PMA promoted the phosphorylation of ERK1/2 with 50% effective concentrations of approximately 0.1 mM and 50 nM, respectively (data not shown).

The relative importance of the cAMP and inositol phosphate/diacylglycerol pathways to hCG-induced ERK1/2 phosphorylation in MA-10 cells was tested using arginine vasopressin (AVP), because this hormone, acting through the endogenous V1 receptor (a Gq/11-coupled receptor) present in MA-10 cells, stimulates the accumulation of inositol phosphates, but not cAMP (20). In the experiments shown in Fig. 2Go we used two different concentrations of AVP and hCG that enhanced inositol phosphate to the same extent (Fig. 2Go, top panel) and measured their effects on cAMP accumulation (Fig. 2Go, middle panel) and ERK1/2 phosphorylation (Fig. 2Go, bottom panel). These data reveal that the concentrations of AVP that result in an increase in inositol phosphate accumulation similar to that induced by the chosen concentrations of hCG do not stimulate the phosphorylation of ERK1/2. Further studies comparing the effects of a selective inhibitor of protein kinase C, bisindolylmaleimide I, and its inactive analog, bisindolylmaleimide V (19), also excluded the potential involvement of protein kinase C on the LHR-provoked phosphorylation of ERK1/2, because neither of these two compounds (used at a concentration of 10 µM) inhibited the hCG-induced phosphorylation of ERK1/2 (data not shown). At these concentrations, however, bisindolylmaleimide I was an effective inhibitor of the PMA-induced phosphorylation of ERK1/2, whereas bisindolylmaleimide V was not (data not shown). These results agree with previous data showing that protein kinase C inhibitors do not block the hCG-induced phosphorylation of ERK1/2 in primary cultures of rat granulosa cells (11).



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Fig. 2. hCG and AVP Stimulate Inositol Phosphate Accumulation in MA-10 Cells, but Only hCG Stimulates cAMP Accumulation and ERK1/2 Phosphorylation

MA-10 cells transiently transfected with the hLHR-wt (1 µg plasmid/35-mm well) were incubated with buffer only or with the indicated concentrations of AVP or hCG. The lengths of the incubations used for inositol phosphate accumulation (top panel), cAMP accumulation (middle panel), and ERK1/2 phosphorylation (bottom panel) were 60, 30, and 30 min, respectively. Each bar represents the mean ± SEM of results obtained in three independent transfections. *, Significantly different (P < 0.05) from cells incubated with buffer only.

 
To further test for the involvement of cAMP in the hCG-induced phosphorylation of ERK1/2 in MA-10 cells, we cotransfected the cells with an expression vector for cAMP phospodiesterase 4D3 (21). As shown in Fig. 3Go this manipulation inhibited the hCG-induced increase in cAMP accumulation and the phosphorylation of ERK1/2 by approximately 75% and 70%, respectively. As epidermal growth factor (EGF) is a potent stimulator of ERK1/2 phosphorylation, but does not stimulate cAMP accumulation in MA-10 cells (22), the overexpression of phosphodiesterase should have no effect on the EGF-induced phosphorylation of ERK1/2. The bottom panel of Fig. 3Go shows that this is indeed the case.



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Fig. 3. Cotransfection of MA-10 Cells with cAMP Phosphodiesterase 4D3 Inhibits the hCG-Induced cAMP Accumulation and ERK1/2 Phosphorylation

MA-10 cells were transiently transfected with hLHR-wt plus an empty vector or hLHR-wt plus an expression vector for cAMP phosphodiesterase 4D3 (each at 1 µg plasmid/35-mm well) as indicated. A, The cells were incubated with buffer only or with 3 nM hCG for 30 min before measuring cAMP. Each bar represents the mean ± SEM of results obtained in three independent transfections. *, Significantly different (P < 0.05) from cells cotransfected with an empty vector and stimulated with hCG. B, The cells were incubated with buffer only, hCG (3 nM) for 30 min, or EGF (2 nM) for 5 min. Only the relevant portions of a representative phospho-ERK1/2 blot are shown. Total ERK1/2 levels did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of ERK1/2 phosphorylation expressed relative to cells cotransfected with an empty vector and incubated with buffer only as determined in three independent transfections.*, Significantly different (P < 0.05) from cells cotransfected with an empty vector and stimulated with hCG.

 
Roles of PKA, Ras, and Rap on hLHR-Induced Phosphorylation of ERK1/2 in MA-10 Cells
The stimulatory effects of cAMP on ERK1/2 phosphorylation in MA-10 cells can be mediated by PKA or by cAMP-dependent guanine nucleotide exchange factors (cAMP-GEFs) (15, 23). The involvement of these divergent pathways was thus addressed using PKA inhibitors and cAMP analogs that selectively activate cAMP-GEFs.

The data summarized in Fig. 4AGo show that overexpression of a specific PKA inhibitor peptide (PKI) (24) impairs the hCG-induced phosphorylation of ERK1/2 by approximately 50%, but overexpression of an inactive form of this inhibitor (PKI-m) has no effect. Similarly, treatment of MA-10 cells with H89, a selective chemical inhibitor of PKA (19) blocked the hCG- induced phosphorylation of ERK1/2 by about 50% (Fig. 4AGo). In addition to using an inactive form of PKI as a negative control, the specificity of the effects of PKI and H89 is documented by the finding that neither of them affected the ability of EGF to stimulate the phosphorylation of ERK1/2 (Fig. 4AGo). In a complementary set of experiments we tested the effects of 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP (8CPT-2Me-cAMP), a cAMP analog that is selective for cAMP-GEFs (23), on ERK1/2 phosphorylation. The results of these experiments (Fig. 4BGo) show that the cAMP-GEF-selective cAMP analog has no effect on ERK1/2 phosphorylation, whereas 8Br-cAMP, a cAMP analog that displays no selectivity for PKA and cAMP-GEFs, robustly stimulates the phosphorylation of ERK1/2.



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Fig. 4. Effects of PKA Inhibitors and cAMP Analogs on the Phosphorylation of ERK1/2 in MA-10 Cells

A, MA-10 cells were transiently transfected with hLHR-wt and an empty vector, hLHR and an expression vector for the wild-type PKI, or hLHR plus an expression vector for an inactive mutant of PKI (PKI-mt; each plasmid was used at 1 µg plasmid/35-mm well). Some of the cells cotransfected with an empty vector were preincubated with buffer only or H89 (50 µM) as indicated. Cells were subsequently incubated with buffer only, hCG (3 nM for 30 min), or EGF (2 nM for 5 min) as indicated. Only the relevant portions of a representative phospho-ERK1/2 blot are shown. Total ERK1/2 levels did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of ERK1/2 phosphorylation expressed relative to cells cotransfected with an empty vector and incubated with buffer only as determined in three independent transfections. a, Significantly different (P < 0.05) from cells cotransfected with an empty vector and stimulated with hCG. b, Significantly different (P < 0.05) from cells cotransfected with an empty vector, preincubated without H89, and stimulated with hCG. B, The levels of phospho-ERK1/2 were ascertained in MA-10 cells that had been incubated with buffer only or the indicated cAMP analogs (0.5 mM for 15 min). Total ERK1/2 levels did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of ERK1/2 phosphorylation expressed relative to cells incubated with buffer only as determined in three independent experiments. *, Significantly different (P < 0.05) from cells incubated with buffer only.

 
Ras and Rap are two guanosine triphosphatases (GTPases) that could potentially act downstream of cAMP in the ERK1/2 activation pathway used by the LHR in MA-10 cells (15, 23). Their involvement was initially tested by measuring the levels of activated Rap1 and Ras in cells treated with hCG or cAMP analogs. The levels of active (i.e. GTP-bound) Ras and Rap were measured by absorption of cell extracts to glutathione-S-transferase (GST) fusion proteins encoding for the Ras binding domain of Raf-1 and the Rap binding domain of Ral guanine nucleotide dissociation stimulator, respectively. As only the active forms of Ras and Rap bind to these domains, visualization of the eluted proteins with Ras or Rap antibodies can be conveniently used to measure the levels of activated Ras or Rap (25, 26). Fig. 5Go shows that hCG does not activate Rap1, but it activates Ras. These data also show that the LHR-induced activation of Ras appears to be mediated by PKA, because it can be inhibited by H89. The same conclusion can be drawn with the use of a selective cAMP analog, because Ras can be activated by 8Br-cAMP, but not by 8CPT-2Me-cAMP (Fig. 5Go). Lastly, the data presented in Fig. 5Go also show that EGF does not activate Rap1, but it activates Ras, and this activation is not inhibited by H89. These findings document the specificity of the inhibitory effect of H89 on the LHR-mediated activation of Ras.



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Fig. 5. Effects of hCG, EGF, cAMP Analogs, and H89 on Ras and Rap1 Activation in MA-10 Cells

MA-10 cells transiently transfected with the hLHR-wt (1 µg plasmid/35-mm well) were preincubated with buffer only or H89 (50 µM) for 30 min and then incubated with buffer only, hCG (3 nM for 15 min), EGF (2 nM for 5 min), 8Br-cAMP (0.5 mM for 30 min), or 8CPT-2Me-cAMP (0.5 mM for 30 min) as indicated. The blots represent the relevant portions of a representative experiment that measured the GTP-bound form of Ras and Rap1 (see Materials and Methods). Total levels of Ras and Rap1 did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of Ras or Rap1 activation expressed relative to cells incubated with buffer only as determined in three independent transfections. a, Significantly different (P < 0.05) from cells preincubated without H89 and stimulated with hCG. b, Significantly different (P < 0.05) from cells incubated with buffer only.

 
Additional functional experiments on the involvement of Ras and Rap were conducted by testing the effects of overexpression of their GTPase-deficient counterparts on the hCG-induced phosphorylation of ERK1/2. Figure 6Go shows that overexpression of a GTPase-deficient mutant of Ras or preincubation of the cells with H89 inhibits the hCG-provoked phosphorylation of ERK1/2 by about 50% and that the combination of these treatments completely prevents the hCG-induced phosphorylation of ERK1/2. Also note that, as expected from the results presented above (c.f. Figs. 4AGo and 5Go), Ras-S17N is an effective inhibitor of the EGF-induced phosphorylation of ERK/12, but H89 is not an effective inhibitor, and it does not potentiate the Ras-S17N-induced inhibition (Fig. 6AGo). In complementary experiments we showed that a GTPase-deficient mutant of Rap1b (Rap1b-S17N) had no effect on the ability of hCG or EGF to provoke the phosphorylation of ERK1/2 (data not shown).



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Fig. 6. Overexpression of GTPase-Deficient Mutant of Ras Inhibits the hCG-Induced Phosphorylation of ERK1/2 in MA-10 Cells

A, MA-10 cells were transiently transfected with hLHR-wt and an empty vector or an expression vector for a GTPase-deficient mutant of Ras (HA-Ras-S17N; each at 1 µg plasmid/35-mm well). The transfected cells were preincubated with or without H89 (50 µM) for 30 min and then incubated with buffer only, hCG (3 nM for 30 min), or EGF (2 nM for 5 min) Only the relevant portions of a representative phospho-ERK1/2 blot are shown. Total levels of ERK1/2 did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of ERK1/2 phosphorylation expressed relative to cells incubated with buffer only as determined in three independent transfections. *, Significantly different (P < 0.05) from cells cotransfected with an empty vector, preincubated without H89, and stimulated with hCG. B, MA-10 cells were transiently transfected with hLHR-wt and an empty vector or an expression vector for a GTPase-deficient mutant of Ras (HA-Ras-S17N; each at 1 µg plasmid/35-mm well) as indicated. Western blots of whole cell lysates were revealed with anti-Ras or anti-HA antibodies as indicated. The endogenous and transfected Ras proteins are indicated by the arrows marked e and t, respectively.

 
These results clearly show that cAMP, PKA, and Ras (but not Rap1) are components of the pathway by which hCG stimulates the phosphorylation of ERK1/2.

Roles of PKA, Ras, and Rap on the cAMP-Induced Phosphorylation of ERK1/2 in Primary Cultures of Immature Rat Leydig Cells
To determine whether the pathway described above applies to normal Leydig cells, we tested the effects of cAMP analogs on Ras activation and ERK1/2 phosphorylation in a progenitor rat Leydig cell model (i.e. primary cultures of Leydig cells isolated from 21-d-old rats) developed by Hardy and colleagues (27, 28, 29, 30). These cells were chosen because they are believed to be good representatives of the precursors of the adult population of mammalian Leydig cells (27, 31).

In agreement with the data obtained with MA-10 cells (see above), we found that 8Br-cAMP, but not 8CPT-2Me-cAMP, induced activation of Ras and phosphorylation of ERK1/2 in primary cultures of progenitor rat Leydig cells (Fig. 7AGo). As an independent confirmation of this finding we also tested the ability of H89 to inhibit the stimulatory effect of 8Br-cAMP. The results presented in Fig. 7BGo show that the 8Br-cAMP-mediated activation of Ras and phosphorylation of ERK1/2 were effectively inhibited by H89. At the same concentration, however, H89 had no effect on the EGF-induced activation of Ras or the phosphorylation of ERK1/2.



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Fig. 7. cAMP Analogs Activate Ras and Stimulate ERK1/2 Phosphorylation in Primary Cultures of Progenitor Rat Leydig Cells in a PKA-Dependent Fashion

A, Primary cultures of progenitor rat Leydig cells were prepared and maintained as described in Materials and Methods and incubated with 8Br-cAMP or 8CPT-2Me-cAMP (0.5 mM) for 15 min. B, Primary cultures of progenitor rat Leydig cells were prepared and maintained as described in Materials and Methods. They were preincubated with buffer only or H89 (50 µM) for 30 min and then incubated with buffer only, 8Br-cAMP (1 mM for 15 min), or EGF (2 nM for 5 min). Only the relevant portions of representative blots depicting the GTP-bound form of Ras or phospho-ERK1/2 are shown. Total levels of Ras or ERK1/2 did not change (data not shown). The numbers at the bottom show a quantitative assessment (mean ± SEM) of Ras activation or ERK1/2 phosphorylation expressed relative to cells incubated with buffer only as determined in three independent experiments. a, Significantly different (P < 0.05) from cells incubated with buffer only. b, Significantly different (P < 0.05) from cells preincubated without H89 and stimulated with 8Br-cAMP.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein-coupled receptor (GPCR)-mediated phosphorylation of ERK1/2 can occur by G protein-dependent and/or -independent pathways (reviewed in Refs. 15, 18 , and 32, 33, 34, 35, 36). The GPCR/G protein-dependent phosphorylation of ERK1/2 can occur by multiple pathways that are mediated by the G{alpha}- or Gß/{gamma}-subunits liberated from the GPCR-promoted activation of different families of G proteins (18, 32, 33). The Gß/{gamma}-mediated pathway is used mostly by GPCRs that couple to the Gi/o family of G proteins, and it involves the Gß/{gamma}-mediated trans-activation of the EGF receptor (37, 38, 39). Although the hLHR activates the Gi/o family of G proteins (8, 40, 41), the activation of this G protein family appears to play a minor role (if any) in the LHR-provoked phosphorylation of ERK1/2. This conclusion is based on the findings that the use of pertussis toxin and sequestrants of Gß/{gamma} induced only a 20–30% inhibition of the hCG-induced stimulation of ERK1/2 phosphorylation (data not shown). In addition, we have not been able to detect enhanced phosphorylation of the EGFR in MA-10 cells treated with hCG or cAMP analogs (data not shown).

G{alpha}s and G{alpha}q/11, which are liberated as a consequence of the activation of the LHR (8, 40, 41), activate second messenger cascades that can also lead to the phosphorylation of ERK1/2 (18). The involvement of G{alpha}q/11-activated second messenger cascades in the LHR-induced phosphorylation of ERK1/2 in MA-10 cells can be readily excluded by three independent sets of observations. First, activation of another Gq/11-coupled GPCR endogenous to MA-10 cells (the V1 arginine vasopressin receptor) does not result in the phosphorylation of ERK1/2. Second, a selective inhibitor of protein kinase C has little or no effect on the hCG-induced phosphorylation of ERK1/2. Third, when engaged by hCG, the low levels of endogenous LHR present in MA-10 cells do not mediate an increase in inositol phosphate accumulation, but they do mediate an increase in cAMP accumulation and ERK1/2 activation (6, 20). This conclusion is in agreement with the data of others showing that inhibitors of protein kinase C do not block the ability of hCG to stimulate ERK1/2 phosphorylation in primary cultures of rat granulosa cells (11).

In contrast, the involvement of the G{alpha}s/adenylyl cyclase/cAMP signaling cascade in the LHR-induced phosphorylation of ERK1/2 in MA-10 cells can be readily documented by overexpression of a cAMP phosphodiesterase to prevent the hCG-induced increase in cAMP accumulation.

The effects of cAMP on the ERK1/2 cascade are complex and cell type specific (reviewed in Ref.15). The actions of cAMP are mediated by PKA (42) or cAMP-GEFs (43, 44), and both of these have been reported to serve as intermediates in the cAMP- induced phosphorylation of ERK1/2 (15, 23). In MA-10 cells and progenitor rat Leydig cells, PKA seems to be the only mediator of cAMP-induced phosphorylation of ERK1/2, however, as judged by the effects of two structurally unrelated inhibitors of PKA and a cAMP analog that selectively activates cAMP-GEFs. Small GTPases, such as Ras, Rap1, and Rap2, have also been implicated as mediators of the actions of cAMP on ERK1/2 phosphorylation. These are located downstream of PKA or the cAMP-GEFs, and they participate in ERK1/2 phosphorylation by virtue of their ability to switch on Raf, the first kinase in the ERK1/2 cascade (15). Our results show that the actions of hCG and cAMP on ERK1/2 phosphorylation in MA-10 cells and primary cultures of rat Leydig cells are mediated by PKA and Ras rather than cAMP-GEFs and Rap.

As mentioned earlier, there have been several reports on the mechanisms by which the LHR may provoke ERK1/2 phosphorylation, but none of these studies has been performed in Leydig cells. The LHR-mediated stimulation of ERK1/2 phosphorylation in a heterologous cell type (COS-7 cells) appears to be mediated by Gß/{gamma} (9). In contrast, studies with primary cultures of porcine (10) or rat granulosa cells (11) as well as human granulosa/luteal cells (10) or with immortalized rat granulosa cell lines expressing the recombinant rat LHR (14) indicate that the LHR-mediated activation of the ERK1/2 cascade in ovarian cells is a cAMP/PKA-dependent process, but the effectors located downstream of PKA were not investigated. In agreement with the data obtained in ovarian cells (10, 11, 14), the data presented here show that the LHR-induced phosphorylation of ERK1/2 in MA-10 cells is meditated by cAMP and PKA. More importantly, however, the data presented here provide novel details about the mechanisms involved in the LHR-induced phosphorylation of ERK1/2. Collectively, our data show that the dominant pathway used by the LHR in Leydig cells involves the activation of Gs, which results in the stimulation of adenylyl cyclase leading to an increase in cAMP, which then activates Ras in a PKA-dependent manner. The activated Ras, in turn, switches on the protein kinase cascade that ultimately results in ERK1/2 phosphorylation. Although we do not know whether the activation of Ras is directly or indirectly mediated by PKA, these results help delineate a cAMP-dependent pathway for ERK1/2 activation that is just beginning to be recognized. Although it has been previously shown that that both Rap1 and Ras can serve as mediators of the ability of cAMP to activate the ERK1/2 cascade in a variety of cell types (reviewed in Ref.15), the PKA dependency of the activation of Ras reported here is novel, because in most cells this event has been reported to be PKA independent (45, 46, 47). In fact, the potential involvement of a PKA-dependent activation of Ras as a mediator of the effects of cAMP on the ERK1/2 cascade was initially recognized as our studies were in progress during the development of cAMP analogs that activate cAMP-dependent guanine nucleotide exchange factors but do not activate PKA (23). A similar conclusion was reached in recent studies of the mechanisms by which other Gs-coupled GPCRs (i.e. the serotonin receptors) activate the ERK1/2 cascade in heterologous cell types (48).

As mentioned above, activation of the LHR is important for the differentiation, proliferation, and perhaps even the transformation of Leydig cells. The data presented here provide novel information regarding the pathway by which the LHR activates a classic mitogenic pathway (49) in Leydig cells. In addition, the LHR-induced, cAMP-mediated phosphorylation of ERK1/2 has been recently implicated as a negative regulator of steroidogenesis in granulosa cells (13, 14), and a similar pathway may very well be operative in Leydig cells. The next challenges will be to determine whether Ras is directly or indirectly activated by PKA and to characterize the involvement of the ERK1/2 cascade in the proliferation of Leydig cells and/or the modulation of their differentiated functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
The preparation of expression vectors (all in the pEF1/V5-His vector from Invitrogen, San Diego, CA) encoding for the hLHR-wt modified with the Myc epitope at the N terminus has been described (6). The origin and handling of MA-10 cells have also been described (16). Transfections were performed in cells plated in 35-mm wells using Lipofectamine as described previously (6), except that the posttransfection 48-h incubation at 30 C was replaced with a 37 C incubation for 24 h. Each well was transfected with 1 µg hLHR expression vector and 1 µg empty vector or 1 µg of the other expression vectors indicated in the figures. An expression vector for cAMP phosphodiesterase 4D3 (50) was donated by Dr. Marco Conti (Stanford University, Stanford, CA), and expression vectors for the PKA inhibitor and its inactive mutant (24) were donated by Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR). An expression vector for the GTPase-deficient (S17N), hemagglutinin (HA)-tagged form of Ras was purchased from the Guthrie Center (Sayre, PA).

Progenitor rat Leydig cells were isolated from 21-d-old male rats as described previously (28, 51). They were allowed to attach to gelatin-coated, 35-mm wells in serum-free medium for 18–24 h (29). At this point the medium was changed to a 1:1 mixture of DMEM/Ham’s F-12 supplemented with 2% newborn calf serum, and the cells were allowed to multiply for 5 d before use. Most of the cells present at the end of this 5-d culture period were positive for 3ß-hydroxysteroid dehydrogenase (data not shown), which is a Leydig cell marker (52).

Kinase Phosphorylation and Ras Activation Assays
MA-10 cells or primary cultures of progenitor rat Leydig cells (all in 35-mm wells) were switched to serum-free medium (Weymouth’s MB752/1 containing, 20 mM HEPES, 50 µg/ml gentamicin, and 1 mg/ml BSA, pH 7.4) for 24 h. At the end of this incubation, single wells were incubated with the appropriate stimuli using the concentrations and times indicated in the figure legends. These were optimized empirically to attain optimal levels of stimulation.

For phosphorylated MEK or ERK assays the cells were placed on ice, the medium was aspirated quickly, and the cells were washed twice with a cold buffer containing 0.15 M NaCl and 20 mM HEPES (pH 7.4) and lysed with 120–200 µl lysis buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and 50 mM Tris-Cl, pH 7.4) by gentle rocking for 30 min at 4 C. The cell lysates were clarified by centrifugation assayed for protein content using the bicinchoninic acid protein assay kit from Bio-Rad Laboratories (Richmond, CA), diluted with either 2x or 5x concentrated sodium dodecyl sulfate gel sample buffer with reducing agents, and boiled for 5 min. Aliquots of the lysates containing identical amounts of protein (30–50 µg) were resolved on 12% SDS-PAGE gels and transferred electrophoretically to polyvinylidene difluoride membranes (53, 54). Phosphorylated ERK1/2 and total ERK1/2 were visualized in the blots during an overnight incubation at room temperature with a phospho-ERK1/2 antibody (E-4, Santa Cruz Biotechnology, Santa Cruz, CA; used at a 1:500 dilution) or a total ERK1/2 antibody (C-14, Santa Cruz Biotechnology; used at a 1:1000 dilution), followed by a 1-h incubation with a secondary antibody covalently coupled to horseradish peroxidase (Bio-Rad; used at a 1:3000 dilution). Phosphorylated MEK1/2 was visualized with a phospho-MEK1/2 antibody (no. 9121, Cell Signaling, Beverly, MA; at a 1:1000 dilution). All immune complexes were ultimately visualized and quantitated using the Super Signal West Femto Maximum Sensitivity system of detection (Pierce Chemical Co., Rockford, IL) and a Kodak digital imaging system (Eastman Kodak Co., Rochester, NY). This image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images.

Ras and Rap1 activation were measured using GST fusion proteins of the Ras binding domain of Raf-1 or the Rap binding domain of Ral to "pull down" the activated (i.e. GTP-bound) forms of Ras and Rap, respectively, which were subsequently detected using Ras and Rap antibodies, respectively. This was performed using kits purchased from Upstate Biotechnology (Charlotsville, VA) as follows. Cells were placed on ice, washed twice with a cold buffer containing 0.15 M NaCl and 20 mM HEPES, pH 7.4, and scraped into 300–800 µl of a lysis buffer containing 25 mM HEPES, 150 mM NaCl, 1% Nonidet P-40, 5 mM MgCl2, 1 mM EDTA, and 2% glycerol, pH 7.5. The cell lysates were clarified by centrifugation, and the protein concentration was equalized after assaying for protein content using the bicinchoninic acid protein assay kit from Bio-Rad. Aliquots of the lysates (200 µg protein in 250 µl for the Leydig cell cultures or 1.2 mg protein in 700 µl for the MA-10 cell cultures) were then mixed with 10- to 20-µl aliquots of GST fusion proteins of the Ras binding domain of Raf-1 or the Rap binding domain of Ral GDS bound to glutathione agarose. After 45 min at 4 C, the agarose beads were pelleted by centrifugation and washed three times with 500 µl of the aforementioned lysis buffer. The washed beads were then resuspended in 35 µl sodium dodecyl sulfate gel sample buffer with reducing agents and boiled for 3 min. Aliquots of the supernatant were resolved on SDS-PAGE gels and transferred electrophoretically to polyvinylidene difluoride membranes as described above. The bound (active) Ras or Rap were then visualized in the blots during an overnight incubation with a Ras monoclonal antibody (clone RAS10, Upstate Biotechnology; used at a 1:1000 dilution) or a Rap1 polyclonal antibody (no. 121, Santa Cruz Biotechnology; used at a 1:200 dilution), followed by a 1-h incubation with a secondary antibody covalently coupled to horseradish peroxidase (Bio-Rad Laboratories; used at a 1:3000 dilution). All immune complexes were ultimately visualized and quantitated using the Super Signal West Femto Maximum Sensitivity system of detection (Pierce Chemical Co.) and a Kodak digital imaging system as described above.

Other Methods
The endogenous Ras and the transfected HA-Ras-S17N were visualized in cell extracts prepared and processed as described above, except that they were developed with a monoclonal antibody to Ras (clone RAS10, Upstate Biotechnology; used at a 1:1000 dilution) or HA (12CA5, Roche, Indianapolis, IN; used at a 1:1000 dilution), followed by a 1-h incubation with a secondary antibody covalently coupled to horseradish peroxidase (from Bio-Rad Laboratories; used at a 1:3000 dilution). Inositol phosphates and cAMP determinations were performed as described previously (6).

Statistical analyses (t test) were performed using the InStat software package from GraphPad, Inc. (San Diego, CA).

Hormones and Supplies
Purified hCG (CR-127; ~13,000 IU/mg) was purchased from Dr. A. Parlow and the National Hormone and Pituitary Agency, NIDDK, and purified recombinant hCG1 was provided by Ares Serono (Randolph, MA). Cell culture medium was obtained from the Cell Production Core, Diabetes and Endocrinology Research Center, University of Iowa (Ames, IA). Other cell culture supplies and reagents were obtained from Corning (Corning, NY) and Life Technologies, Inc. (Gaithersburg, MD), respectively. 8Br-cAMP, PMA, AVP, pertussis toxin, and recombinant EGF were obtained from Sigma-Aldrich Corp. (St. Louis, MO). H89, bisindolylmaleimide I, and bisindolylmaleimide V were obtained from Calbiochem (La Jolla, CA), and U0126 and 8CPT-2Me-cAMP were purchased from Tocris Neuramin (Bristol, UK). All other chemicals were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We thank Drs. Marco Conti (Stanford University), and Richard Maurer (Oregon Health Sciences University) for providing us with several constructs used here. We also thank Ares Serono for providing us with the original hLHR construct and recombinant hCG, and Drs. Deborah Segaloff and Colette Galet for reading the manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant CA-40629 (to M.A.) and a fellowship from the Lalor Foundation (to T.H.). The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by NIH Grant DK-25295.

1 Both preparations were used in this study and were found to be indistinguishable. Back

Abbreviations: AVP, Arginine vasopressin; 8Br-cAMP, 8-bromo-cAMP; cAMP-GEF, cAMP-dependent guanine nucleotide exchange factor; 8CPT-2Me-cAMP, 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP; EGF, epidermal growth factor; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; GTPase, guanosine triphosphatase; HA, hemagglutinin; hCG, human choriogonadotropin; hLHR, human LH receptor; PKA, protein kinase A; PKI, protein kinase A inhibitor peptide; PMA, phorbol 12-myristate 13-acetate; wt, wild-type.

Received for publication June 2, 2003. Accepted for publication August 5, 2003.


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