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Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells
European Journal of Pharmacology 691 (2012) 283–291
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells Aline Rosa Nascimento, Maristela Taliari Pimenta, Thais F.G. Lucas, Carine Royer, Catarina Segreti Porto, Maria Fatima Magalhaes Lazari n ~ Paulo, Sao ~ Paulo, Brazil Section of Experimental Endocrinology, Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de Sao
a r t i c l e i n f o
abstract
Article history: Received 30 January 2012 Received in revised form 28 June 2012 Accepted 3 July 2012 Available online 20 July 2012
Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of Gi. AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24 h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was Gi- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to Gi and activation of both MEK/ERK1/2 and PI3K/AKT pathways. & 2012 Elsevier B.V. All rights reserved.
Keywords: Relaxin RXFP1 Sertoli cell Proliferation ERK1/2 PI3K/AKT
1. Introduction Relaxin is a member of the family of insulin-related peptides, first recognized for its important role during pregnancy and parturition, but now known to have several additional functions (Sherwood, 2004; Dschietzig et al., 2006). Relaxin is structurally similar to insulin, but binds to RXFP1 (relaxin family peptide receptor 1), a G-protein coupled receptor (GPCR) that belongs to the subfamily of leucine-rich repeat containing GPCRs (LGRs), which also includes the receptors for the glycoprotein hormones TSH, FSH and LH (Hsu et al., 2002). The role of relaxin in male reproduction is still unclear (Ivell et al., 2011). It was initially thought that relaxin was produced by the prostate and released to the seminal fluid to affect sperm motility (Sasaki et al., 2001; Sherwood, 2004). More recently, relaxin has been shown to influence sperm functionality (Ferlin et al., 2011; Miah et al., 2011). Although the major production of relaxin in the male reproductive tract occurs in the prostate,
n
Corresponding author. Fax: þ5511 5576 4448. E-mail addresses: [email protected] (A.R. Nascimento), [email protected] (M.T. Pimenta), [email protected] (T.F.G. Lucas), [email protected] (C. Royer), [email protected] (C.S. Porto), [email protected] (M.F.M. Lazari). 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.021
testes are also a source of relaxin (Cardoso et al., 2010; Gunnersen et al., 1995; Kohsaka et al., 2009). Studies with relaxin gene knockout animals (Rln / ) revealed that relaxin plays a role in the growth and development of the male reproductive tract (Samuel et al., 2003). The Rln / mice have smaller testis, decreased sperm maturation, and increased apoptosis (Samuel et al., 2003, 2005). Relaxin mRNA levels are higher in the testis of immature than adult rats, and Sertoli cells of immature rats represent an important source of relaxin mRNA (Cardoso et al., 2010). Relaxin induces proliferation of cultured rat Sertoli cells, suggesting an autocrine or paracrine role (Cardoso et al., 2010; Filonzi et al., 2007). Sertoli cells play an essential role in several stages during life. They are important for the expression of the male phenotype and to support spermatogenesis during puberty (Sharpe et al., 2003). The number of Sertoli cells will determine the extension of sperm production. There are several evidences that interplay between Ras/Raf/ MEK/ERK and PI3K/AKT pathways may be necessary for normal cell proliferation to occur (rev. Chambard et al., 2007), and both pathways are important for Sertoli cell function (Meroni et al., 2002; Cheng et al., 2007; Fix et al., 2004; Royer et al., 2012). GPCR agonists may promote cell proliferation and differentiation through the activation of these pathways (rev. Rozengurt, 2007).
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Ras/Raf/MEK/ERK and PI3K/AKT pathways may be activated by Gas, Gaq or bg subunits released from Gi or G0 (Yart et al., 2002; Stork and Schmitt, 2002). GPCRs may also activate the ERK1/2 and PI3K/AKT pathways through the transactivation of growth factor receptors (Schlessinger, 2000; Ohtsu et al., 2006; Rozengurt, 2007; Lucas et al., 2008), and through G protein-independent and arrestin-dependent mechanisms (Lefkowitz and Shenoy, 2005). The mechanism by which relaxin stimulates proliferation and differentiation is not yet clear. When overexpressed in HEK293T cells, RXFP1 couples with Gs, G0B, and Gi3, affecting cyclic AMP accumulation in a complex manner (Halls et al., 2006, 2007). Endogenous expression of RXFP1 allows a more selective coupling to G proteins, depending on the cellular context (rev. in Halls, 2012). This study investigated the pathways involved in the relaxin-stimulated proliferation of rat Sertoli cells, which endogenously express RXFP1 (Filonzi et al., 2007).
2. Material and methods 2.1. Primary cell culture The experimental procedures were approved by the Research Ethical Committee from UNIFESP-EPM (CEP0001/09). We used 15-day old male Wistar rats that were housed in the Animal Facility at the Instituto de Farmacologia e Biologia Molecular (INFAR), Escola Paulista de Medicina-Universidade Federal de Sa~ o Paulo (EPM-UNIFESP), and maintained on a 12 h light/12 h dark lighting schedule at 23 1C, with food and water freely available. The testes were removed and decapsulated, and Sertoli cells were isolated as previously described (Grima et al., 1995; Lucas et al., 2004, 2008). Cells were plated at a density of approximately 4 106 cells/100 mm dish (about 5 104 cells/ cm2), in phenol-red free Ham’s F12/Dulbecco’s Modified Eagle Medium (F12/DMEM 1:1, Gibco, Invitrogen, Grand Island, NY, USA) containing 0.02 g/l gentamicin (Sigma Chemical Co., St Louis, MO, USA), pH 7.2–7.4, and supplemented with 10 mg/ml insulin, 10 mg/ml transferrin, 10 ng/ml sodium selenite and 10 ng/ml epidermal growth factor (Sigma). Cells were grown in a humidified atmosphere of 5% CO2–95% air at 35 1C for 48 h, treated with 20 mM Tris–HCl, pH 7.4, to lyse residual germ cells (Galdieri et al., 1981), and allowed to grow for another 24 h. The culture was analyzed by morphological and immunocytochemical techniques (Lucas et al., 2008), confirming that the large majority of the population was Sertoli cells. Culture medium was replaced by another one without supplements 20 h before the experiments. At this stage, cells were 90–95% confluent and the viability, as determined by trypan blue exclusion, was higher than 90%. For proliferation assays, Sertoli cells were prepared as described above, plated at a low density, and at the day of experiments the confluence was 50–60%. 2.2. [Methyl-3H] thymidine incorporation assays Incorporation of [methyl-3H] thymidine into cell DNA was estimated as described by Guizzetti et al. (1996). Previous studies in our laboratory indicated that incorporation of [methyl-3H]thymidine (2 mCi/ml, specific activity 79.0 Ci/mmol, GE) in cultured Sertoli cells was time-dependent and linear from 2 to 10 h of incubation (Lucas et al., 2004). Therefore, primary Sertoli cell cultures on culture day 4 were initially incubated with 2 mCi/ml [methyl-3H]thymidine for 6 h at 35 1C. Afterwards, cells were incubated in the absence or presence of the MEK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2aminophenylthio]-butadiene (U0126, 20 mM, 30 min, Cell Signaling Technology, Danvers, MA), the PI3K inhibitor wortmannin (100 nM,
30 min, Sigma), the PKA inhibitor N-[2-[[3-(4-bromophenyl)-2propenyl]amino]ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 2 mM, 2 h, Sigma), the selective inhibitor of EGFR kinase, 4-(3chloroanilino)-6,7-dimethoxyquinazoline (AG1478, 1 mM, 15 min, Calbiochem., San Diego, CA, USA) or the PKC inhibitor GF 109203X (5 mM, 30 min, Sigma). Incubation was continued in the absence or presence of these inhibitors and in the absence or presence of recombinant human relaxin H2 (RLN; Phoenix Pharmaceuticals Co., Burlingame, CA, USA), 50 ng/ml, for 24 h at 35 1C. The reaction was stopped by cooling the cells at 0 1C. The medium was aspirated and the cells rinsed twice with ice-cold PBS and 5% trichloroacetic acid (Sigma). The cells were then solubilized with 0.5 N NaOH, collected with cotton-swabs, and transferred to 5 ml OptiPhase HiSafe-3 scintillation liquid (PerkinElmer Life Science Products, Boston, MA, USA). Bound radioactivity was determined in a scintillation b counter (LS 6000 IC, Beckman Colter Inc., Palo Alto, CA, USA). Results were expressed in relation to control, basal levels of [methyl-3H] thymidine incorporation (absence of relaxin and inhibitors) 2.3. Determination of cadherins 1 and 2 mRNA levels by real-time qRT-PCR 2.3.1. RNA extraction and cDNA synthesis Cells were incubated with 50 ng/ml of relaxin at 35 1C for 2 and 4 h. The reaction was stopped on ice, and cells were washed with cold sterile phosphate-buffered solution (PBS). Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the standard protocol (Chomczynski and Sacchi, 1987). RNA concentration was measured by spectrophotometry, and OD 260:280 nm ratios between 1.8 and 2.1 were obtained for all RNA samples. Ribosomal RNA integrity was checked by agarose gel electrophoresis, and a sharp and clear 2:1 ratio of ethidium bromide stained 28S:18S rRNA bands was observed for all samples. Two micrograms total RNA were used to synthesize the first strand cDNA at 50 1C with the Superscript III first strand synthesis Supermix (Invitrogen) and the oligo-dT primer supplied with this kit. 2.3.2. Quantitative PCR For the qPCR, we used the SYBR Green system (Applied Biosystems, Foster City, CA, USA). The following oligonucleotides were used to amplify fragments of cadherin 1 (Cdh1), cadherin 2 (Cdh2) and the endogenous control b-actin (Actb): Cdh1: 50 ACCGGCATCACCACAGAGACC-30 (forward) and 50 - CCGGGCAGTTGATGG GAGGG-30 (reverse); Cdh2: 50 -GCGGCCTTGCTTCAGGCATC30 (forward) and 50 -CTGGCCTTCGTGCACGTCCT-30 (reverse); Actb: 50 - GTAGCCATCCAGGCTGTG TT-30 (forward) and 50 -CCCTCATAGATGGGCACAGT-30 (reverse). The primers (Invitrogen) were designed with the Primer3 program (Rozen and Skaletsky, 2000) and spanned exon–exon boundaries whenever possible. Primers for target genes and beta-actin were designed to have approximately the same amplification efficiency (¼1). Controls without cDNA or without primers were included in each assay. Samples were run in a 7500 Real-Time PCR System (Applied Biosystems), using default conditions of amplification (50 1C for 2 min, 95 1C for 10 min and 40 cycles of 95 1C for 15 s and 60 1C for 1 min). The dissociation curves were obtained at the end to confirm specificity of the amplification. Each sample was run in triplicate. The average cycle threshold (CT) was determined with Applied Biosystems software. Data were analyzed by the comparative DDCT method (ABI PRISM User Bulletin #2, Applied Biosystems). The control values were always used as a calibrator in each experiment. Data are expressed as mean 7S.E.M. of the 2-DDCT from 3 different cDNAs (from three different cultures). At the end of the experiments,
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samples were run in 2% agarose gel electrophoresis to further confirm the absence of nonspecific amplification. The size of the expected products was compared to a DNA ladder (100 bp ladder, Invitrogen).
2.4. Western blot analysis On culture day 4, Sertoli cells were incubated in the absence (control) and presence of 25 or 50 ng/ml of relaxin, for the indicated times at 35 1C. In another series of experiments, before treatment with relaxin the cells were untreated or pretreated at 35 1C with the MEK1/2 inhibitor U0126 (20 mM, 30 min), the PI3K inhibitor wortmannin (100 nM, 30 min), the PKA inhibitor H89 (2 mM, 2 h), the Gi inhibitor pertussis toxin (PTX, 100 ng/ml, 16 h; Sigma), or the SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min; Calbiochemical, San Diego, CA, USA). At these concentrations, the inhibitors are highly selective, as previously reported in Sertoli and other cells (Cre´pieux et al., 2001; Lucas et al., 2008, 2010; Halls et al. 2006; Tai et al., 2009; Moore et al., 2007; Peters et al. 1999). Medium was removed, the cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, 10 mM Na4P207), as previously described (Lucas et al., 2008; Royer et al., 2012). Protein concentration was determined with a BioRad protein assay, using BSA as standard (Bio Rad Laboratories, Hercules, CA, USA).
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Total cellular proteins (30 mg/lane for AKT, and 60 mg/lane for ERK1/2 and PCNA) were incubated with sample buffer containing b-mercaptoethanol and subjected to SDS/PAGE. Proteins were electrotransferred onto PVDF membranes overnight, 20 V at 4 1C. Membranes were blocked in TBS containing 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk, pH 7.6, for 2 h at room temperature. Membranes were washed in TBS-T, and incubated overnight at 4 1C with primary antibody. To mark total and phosphorylated MAP kinase, we used rabbit polyclonal antibodies against rat p44/ p42 MAP kinase (#9102, Cell Signaling Technology), and phospho-p44/p42 MAP kinase (Thr202/Tyr204, #9101, Cell Signaling Technology) diluted 1:1000 and 1:2000, respectively, in blocking solution. Total and phosphorylated AKT were labeled with rabbit monoclonal antibody against a fragment in the C-terminal sequence of mouse AKT (pan-AKT, Cell Signaling Technology), and polyclonal antibody against phospho-Ser473 AKT (p-AKT, Ser473, Cell Signaling Technology), diluted in blocking solution, 1:2000 and 1:1000, respectively. Proliferating cell nuclear antigen (PCNA) was detected with a mouse monoclonal antibody (#2586, Cell Signaling Technology, 1:200). Membranes were next incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (Amersham Biosciences), and proteins were visualized with enhanced chemiluminescence reagent (ECL, GE Healthcare, Piscataway, NJ, USA). Apparent molecular weights were compared with molecular weight standards (New England Biolabs, Boston, MA, USA), and band intensities were quantified by densitometric analysis of linear-range immunoblots by using Epson Expression 1680 scanner (Epson America Inc., CA, USA) and
Fig. 1. Effects of relaxin on proliferation and differentiation of rat Sertoli cells. Effect of relaxin (A) and the inhibitors (B) on Sertoli cell proliferation. Cells were loaded with [methyl-3H]thymidine for 6 h at 35 1C, treated with U0126 (20 mM, 30 min), wortmannin (100 nM, 30 min), H89 (2 mM, 2 h), AG1478 (1 mM, 15 min), or GF109203X (5 mM, 30 min), and incubated in the absence (basal incorporation, control) or presence of relaxin (RLN, 50 ng/ml) for 24 h. Bound radioactivity was determined, and the results were expressed in relation to control (mean 7 S.E.M. of 3–4 independent experiments performed in triplicate). Basal incorporation of [methyl-3H] thymidine was 1440.7 7 164.4 dpm/well, and it was only affected by the PKC inhibitor GF 109203X (Fig. 1B). The asterisks indicate significant difference from control (ANOVA followed by Dunnett’s test, Po 0.05). (C) Western blot analysis of effect of relaxin (RLN, 50 ng/ml for 24 h) on the expression of the proliferating cell nuclear antigen, PCNA. The bar graph shows the densitometric analysis of the Western blots. Results were normalized to GAPDH expression and plotted in relation to control ( ¼ 1). Data are representative of 3 independent experiments. Error bars indicate S.E.M. * Po 0.05, Student’s t test. (D) Real-time PCR analysis of the gene expression of cadherins 1 (Cdh1) and 2 (Cdh2) after stimulation with RLN (50 ng/ml) for 2 and 4 h. Values were normalized using b-actin as endogenous control, and are expressed as relative values (means7 S.E.M.) of 3 independent experiments performed in triplicate. Differences between the three groups were not statistically significant (ANOVA).
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Quick Scan 2000 WIN software (Helena Laboratories, TX, USA). Results were normalized based on total ERK1/2, AKT or GAPDH expression and plotted (mean7S.E.M.) in relation to control, C (¼1). 2.5. Statistical analysis Data were expressed as mean 7S.E.M. Statistical analysis was performed with GraphPad Prism 5, version 5.01 (GraphPad Software Inc.). ANOVA was followed by Newman–Keuls for multiple comparisons, or Dunnett’s test for comparison with control. Student’s t-test was used for comparisons between two groups. P valueso0.05 were accepted as significant.
3. Results 3.1. Pathways involved in the relaxin-induced Sertoli cell proliferation Treatment of Sertoli cells with 50 ng/ml relaxin induced a 41% increase of cell proliferation (Fig. 1A), and almost doubled the expression of PCNA (Fig. 1C), which is an auxiliary protein of DNA polymerase d that serves as a marker for proliferation in most cells (Bravo et al., 1987). To determine the pathways involved in the relaxin-induced proliferation of rat Sertoli cells, we analyzed the effect of relaxin in the presence of specific inhibitors of several
pathways: the MEK1/2 inhibitor U0126, the PI3K inhibitor wortmannin, the PKA inhibitor H89, the inhibitor of EGFR activity AG1478, and the PKC inhibitor GF109203X. Relaxin-induced [3H]thymidine incorporation was inhibited by U0126, wortmannin, and H89, but not by GF109203X and AG1478. The inhibitors alone did not significantly affect the basal level of [3H]-thymidine incorporation, except GF109203X, which significantly increased Sertoli cell basal [3H]-thymidine incorporation (Fig. 1B). We also investigated whether relaxin affects the expression of cadherins 1 and 2, which are the constituents of the adherens junctions in testis, and markers of Sertoli cell differentiation (Siu and Cheng, 2004). Treatment of Sertoli cells with relaxin for 2 and 4 h failed to stimulate gene expression of cadherins 1 and 2 (Fig. 1D). 3.2. Relaxin stimulates a Gi-dependent MEK/ERK1/2 phosphorylation in rat Sertoli cells Treatment of primary culture of rat Sertoli cells with relaxin caused a time and concentration-dependent activation of ERK1 and ERK2 phosphorylation (Fig. 2A and B). The activation was rapid and transient, with a peak at 5 min and a complete return to basal control levels after 30 min (Fig. 2A). Based on these results, all subsequent experiments on relaxin-induced ERK1/2 phosphorylation were performed with 5 min incubations with 50 ng/ml relaxin. In addition, since relaxin induced similar activation of ERK1 and ERK2, only the combined phosphorylation of ERK1 and
Fig. 2. Relaxin activates a MEK-dependent ERK1/2 phosphorylation in a time and concentration-dependent fashion. (A) Western blot analysis of the time course of ERK1/2 phosphorylation by 50 ng/ml relaxin. (B) Concentration-dependent phosphorylation of ERK1/2 by 5 min incubation with relaxin. (C) Effect of the MEK1/2 inhibitor U0126 (20 mM, 30 min) on ERK1/2 phosphorylation induced by relaxin (50 ng/ml). (D) Effect of various inhibitors on basal ERK1/2 phosphorylation: MEK1/2 inhibitor U0126 (20 mM, 30 min), PI3K inhibitor wortmannin (100 nM, 30 min), PKA inhibitor H89 (2 mM, 2 h), Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h), and SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min). The bar graphs show the densitometric analysis of the Western blots. (A) and (B) Open bars: ERK1; solid bars: ERK2. (C) and (D) Combined expression of ERK1 and ERK2. Results were normalized to total ERK1/2 expression and plotted in relation to control ( ¼ 1). Error bars show S.E.M., * Po 0.05 (Student’s t test), n¼3–5 independent experiments. Different letters indicate differences among experimental groups (ANOVA and Newman–Keuls test, P o0.05).
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ERK2 is presented. Fig. 2C shows that relaxin-induced ERK1/2 phosphorylation was blocked by the MEK inhibitor U0126. Fig. 2D shows that U0126 and the several other inhibitors used in the subsequent experiments did not significantly affect the basal level of ERK1/2 phosphorylation. RXFP1 can couple with Gs, G0 and Gi (Halls et al., 2012), but Sertoli cells do not seem to express G0 (Paulssen et al., 1991). We investigated the involvement of Gs-cyclic AMP-PKA and Gi on the relaxin-induced ERK1/2 phosphorylation. Fig. 3A illustrates that pre-treatment of cells with the PKA inhibitor H89 reduced the effect of relaxin on ERK1/2-activation. Pretreatment of cells with 100 ng/ml pertussis toxin for 16 h completely blocked the effect of relaxin on ERK1/2 phosphorylation (Fig. 3B). The phosphorylation of ERK1/2 was inhibited by PP2, an inhibitor of the SRC-family of nonreceptor tyrosine kinases (Fig. 3C). In addition, activation of PI3K seems to contribute to relaxin-induced ERK1/2 activation, because pre-incubation with wortmannin blocked this effect (Fig. 3D).
3.3. Relaxin stimulates a Gi-dependent activation of PI3K/AKT pathway Considering that inhibition of PI3K inhibited relaxin-induced ERK1/2 phosphorylation, we investigated in more details the role of relaxin on the activation of PI3K/AKT pathway, measuring the ability of the hormone to induce phosphorylation of the
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AKT-Ser243 (Fig. 4). Fig. 4A shows that only wortmannin significantly affected the basal phosphorylation of AKT. A rapid increase in AKT phosphorylation was observed after 5 min incubation with relaxin, and this effect was completely blocked by pertussis toxin but not by H89 (Fig. 4B). Relaxin-induced phosphorylation of AKT was also completely blocked by the PI3K inhibitor wortmannin and by the MEK inhibitor U0126 (Fig. 4C). It is worth to mention that even after correcting the inhibitory effect of wortmannin on basal phosphorylation of AKT, this inhibitor was still able to completely block relaxin-induced AKT phosphorylation. The relaxin-induced activation of the PI3K/AKT pathway could still be detected after incubation with relaxin for 24 h (results not shown).
4. Discussion We have previously suggested that relaxin plays an autocrine/ paracrine role in testis (Cardoso et al., 2010; Filonzi et al., 2007). The use of pharmacological tools to inhibit specific signaling steps allowed us to suggest that both the MEK/ERK1/2 and the PI3K/ AKT pathway contribute to relaxin-induced Sertoli cell proliferation, and activation of a Gi protein and a SRC nonreceptor tyrosine kinase are the major upstream pathways to activate MEK1/2/ ERK1/2 and PI3K/AKT pathways (Fig. 5). The signaling mechanism underlying the mitogenic effect of relaxin seems to depend on the cell-type. In the prostate cancer
Fig. 3. Pathways involved in the relaxin-induced ERK1/2 phosphorylation. Western blot analysis of expression of phospho-ERK1/2 (top) and total ERK1/2 (bottom), in the absence (control) or presence of relaxin (RLN, 50 ng/ml for 5 min), after pre-incubation with (A) the PKA inhibitor H89 (2 mM, 2 h); (B) the Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h); (C) the SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min); and (D) PI3K inhibitor wortmannin (100 nM, 30 min). All lanes were run on the same gel, but irrelevant lanes were removed from the figure. The bar graphs show the combined expression of ERK1 and ERK2, quantified by densitometric analysis of the Western blots. Results were normalized to total ERK1/2 expression and plotted in relation to control ( ¼1). Error bars indicate S.E.M., n¼ 3–5 independent experiments. Different letters indicate differences among experimental groups (ANOVA and Newman–Keuls, Po 0.05).
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Fig. 4. Relaxin induces AKT (Ser473) phosphorylation in the Sertoli cells. (A) Densitometric analysis of the effect of the inhibitors on basal AKT phosphorylation: MEK1/2 inhibitor U0126 (20 mM, 30 min), PI3K inhibitor wortmannin (100 nM, 30 min), PKA inhibitor H89 (2 mM, 2 h), and Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h). * different from control (ANOVA, followed by Dunnett’s test; P o0.05). (B) Effect of PTX and H89 on AKT (Ser473) phosphorylation induced by relaxin (RLN, 50 ng/ml) for 5 min, n¼2 independent experiments. (C) Effect of wortmannin and UO126 on AKT (Ser473) phosphorylation induced by relaxin (RLN, 50 ng/ml) for 5 min, n ¼3–4 independent experiments. The bar graphs show the densitometric analysis of the immunoblots. Results were normalized to total AKT expression and plotted in relation to control (¼ 1). Error bars indicate S.E.M. Different letters indicate differences between groups (ANOVA and Newman–Keuls test, P o 0.05).
Fig. 5. A model of the intracellular signaling pathways involved in the mitogenic effect of relaxin in Sertoli cells. Relaxin (RLN) binds to the RXFP1 receptor and induces the coupling of the receptor to the Gi protein. Released bg subunits activate a nonreceptor tyrosine kinase of the SRC-family, and the PI3K/AKT pathway, which may stimulate cell proliferation. SRC and PI3K activate the Ras/ Raf/MEK1/2/ERK1/2 pathway, which also contributes to cell proliferation. The site of action of inhibitors is indicated by the blunted arrows. In this model, wortmannin inhibits the PI3K-mediated activation of Ras/MEK/ERK1/2 pathway, and the MEK inhibitor U0126 may inhibit the PI3K/AKT pathway through the release of the activity of the phosphatase and tensin homolog (PTEN), an endogenous inhibitor of PI3K.
cell line LNCaP, relaxin is able to activate both Gas/cyclic AMP/ PKA and PI3K/AKT pathways to induce NFkB and co-translocation of AR and b-catenin to the nucleus (Vinall et al., 2011). Relaxininduced proliferation of cervical epithelial and stromal cells depends on stromal expression of the estrogen receptor ERa, which binds to estrogen response elements (Yao et al., 2010). In Sertoli cells of the rat, relaxin caused a rapid and transient increase of ERK1/2 phosphorylation, peaking after 5 min of
exposure to relaxin, and returning to basal levels after 30 min of exposure. Similar rapid relaxin-induced ERK1/2 activation was seen in human endometrial stromal cells, in the human acute monocytic leukemia cell line THP-1, in smooth muscle cells from the coronary artery, and in cells from human endometrial stroma (Zhang et al., 2002). However, this effect may be cell type dependent, because the relaxin-induced ERK1/2 activation in rat myofibroblasts involved both a rapid (2 min) and a lower but sustained (50 min) component (Mookerjee et al., 2009), and in endothelial cells of umbilical vein and in the cervical cancer human cells HELA, relaxin stimulated the ERK1/2 pathway only after a much longer period of stimulation (45–90 min) (Dschietzig et al., 2003). The PKA inhibitor H89 seemed to affect relaxin-induced Sertoli cell proliferation, and this inhibitor reduced relaxin-induced ERK1/2 phosphorylation, suggesting that the classical Gs/cyclic AMP/PKA pathway described for relaxin in other systems may contribute to the activation of the MEK/ERK1/2 pathway in Sertoli cells. Nevertheless, using an immunoenzymatic assay, we detected a robust accumulation of cyclic AMP in Sertoli cells induced by FSH or forskolin, but not by relaxin (unpublished results). We cannot exclude the possibility that relaxin stimulates a compartmentalized cyclic AMP production in Sertoli cells (Ostrom and Insel, 2004), which could not be easily detected by the immunoenzymatic assay in whole cell extract. ERK1/2 activation was sensitive to pertussis toxin, indicating a Gi-dependent mechanism, and to wortmannin, indicating a PI3Kdependent mechanism. It is well known that GPCRs can activate PI3K through the release of bg subunits from Gi and G0 (Curnock et al., 2002; Yart et al., 2002). Since Sertoli cells do not express G0 (Paulssen et al., 1991), bg subunits released from Gi are probably responsible for the relaxin-induced activation of PI3K. Activation of PI3K can activate the MEK/ERK1/2 pathway by directly phosphorylating Ras (Hawes et al., 1996) or SRC (Rameh et al., 1995). The activation of ERK1/2 by relaxin involved a SRC-dependent mechanism because it was inhibited by the SRC inhibitor PP2. Activation of SRC by relaxin could be mediated by bg subunits of Gi (Liebmann, 2011; Prenzel et al., 1999), or by upstream stimulation
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of PI3K, which generates PtdIns (3,4,5) P3 that is able to bind directly to the SH2 domain of SRC (Rameh et al., 1995). SRC mediates the phosphorylation of Ras, but may also phosphorylate Tyr-845 of the tyrosine kinase domain of EGFR and lead to ERK1/2 activation. However, the mitogenic effect of relaxin is not inhibited by AG1478, an inhibitor of the tyrosine kinase activity of EGFR. PI3K acts on phosphatidylinositols (PtdIns) from the plasma membrane to release (PtdIns)-3,4-P2 (PIP2) and PtdIns-3,4,5-P3 (PIP3). PIP3 activates the novel PKC types e, Z and d (sensitive to GF109203X) (Toker et al., 1994). Although PKC may phosphorylate Ras and activate the Raf/MEK/ERK cascade, the PKC inhibitor GF109203X did not inhibit relaxin-induced Sertoli cell proliferation. In fact, this inhibitor tended to increase cell proliferation, which could be caused by an inhibition of the expression of proteins involved in cell cycle by PKC (Livneh and Fishman, 1997). In conclusion, activation of PKC does not seem to contribute to the mitogenic effect of relaxin in Sertoli cells of the rat. Relaxin-induced AKT phosphorylation, like relaxin-induced ERK1/2 activation, involved the upstream activation of Gi, because it was blocked by pertussis toxin. The activation of the PI3K/AKT pathway by relaxin did not involve upstream activation of the Gs/ cyclic AMP/PKA pathway, because the PKA inhibitor H89 did not affect AKT phosphorylation. Basal phosphorylation of ERK1/2 and AKT was detected even in cells starved from medium supplementation for 20 h. This basal phosphorylation of AKT and ERK1/2 could be due to the action of growth factors, or other autocrine/paracrine factors produced by Sertoli cells, including relaxin. Nevertheless, only wortmannin significantly reduced basal AKT phosphorylation, and the inhibitors did not significantly affect the basal level of ERK1/2 phosphorylation or 3H-thymidine incorporation. Both ERK1/2 and PI3K pathways appear to stimulate Sertoli cell proliferation, and both pathways seem to modulate the activation of each other. This conclusion was based on the observations that AKT phosphorylation was inhibited by the MEK1/2 inhibitor U0126, and ERK1/2 phosphorylation was inhibited by the PI3K inhibitor wortmannin. Similarly, the adipogenic effect of the peroxisome proliferator-activated receptor g (PPARg) also involved activation of PI3K/AKT by ERK1/2 (Chuang et al., 2007). The activation of ERK1/2 pathway may lead to the phosphorylation and inhibition of the phosphatase and tensin homolog, PTEN (Marino et al., 2003), a phosphatase that dephosphorylates PIP3 and inhibits activation of AKT. The modulation of MEK/ERK1/2 activation by PI3K/AKT pathway may be explained not only by the PI3K-mediated activation of Ras, but also because AKT and the phosphoinositide-dependent kinase 1 (PDK1) may phosphorylate and activate the p21-activated kinase-2 (PAK-2), which phosphorylates Raf-1, and consequently leads to the activation of the MEK/ERK1/2 pathway (Siu et al., 2005). FSH and testosterone are important regulators of Sertoli cell function (Shupe et al., 2011). In primary culture of Sertoli cells isolated from 20-day old rats, FSH stimulates adenylyl cyclase and cyclic AMP production via activation of Gs, and PI3K pathway via activation of Gi (Meroni et al., 2002). The FSH-induced activation of ERK1/2 pathway depends on the dual coupling of the FSH receptor with Gs and Gi, in a PKA- and SRC-dependent manner (Cre´pieux et al., 2001). The activation of ERK1/2 by FSH is dependent on the developmental stage of Sertoli cells. FSH stimulates ERK1/2 phosphorylation and proliferation of Sertoli cells from immature rats, but inhibits the ERK pathway when the differentiation of Sertoli cells starts (Cre´pieux et al., 2001). Relaxin and FSH seem to use similar upstream signaling pathways to activate PI3K/AKT and MEK/ERK1/2 pathways, but whether relaxin also stimulates ERK1/2 phosphorylation in differentiating Sertoli cells still remains to be determined. It remains also to be determined whether relaxin plays a role in Sertoli cell
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differentiation. Although relaxin stimulates cadherin 2 expression in MCF7 breast cancer cells and uterus (Sacchi et al., 1994; Ryan et al., 2001), relaxin did not affect gene expression of cadherins 1 and 2 in Sertoli cells. It is well known that the Ras/Raf/MEK/ERK and PI3K/AKT signaling cascades affect cell cycle regulation and cell proliferation. Several ERK targets that affect cell proliferation have been identified, and full induction of some cell cycle players sometimes requires activation of both the MEK/ERK and the PI3K/AKT pathways (rev. in Chambard et al., 2007). We still need to explore in more detail the pathways that follow the relaxin-mediated activation of PI3K/AKT and MEK/ERK1/2 pathways, but activation of transcription factors such as Elk-1 and c-Fos, or a direct effect of PI3K on the expression of proteins that affect cell cycle may be involved (Chang et al., 2003). Relaxin increased proliferation and the expression of PCNA in cultured leiomyoma cells but not in normal myometrial cells (Suzuki et al., 2012). Treatment of immature Sertoli cells with relaxin for 24 h also increased expression of PCNA, which is essential for the S-phase of the cell cycle and represents an important index of cell proliferation. Phosphorylation of the cell cycle inhibitor p21 by PI3K/AKT pathway releases PCNA from the complex p21/cdk1/cdk2/cyclins, allowing its association with DNA polymerase d and the transcription complex AP-1, inducing DNA synthesis and reducing ¨ inhibition of Cdk2 by p21 (Rossig et al., 2001). In conclusion, relaxin activates the PI3K/AKT and MEK-ERK1/2 pathways to induce proliferation of Sertoli cells. We propose the following sequence of events for the mitogenic effect of relaxin (Fig. 5): (1) Relaxin binds to RXFP1 and induces coupling to Gi; (2) released bg subunits activate PI3K and SRC; (3) PI3K activation results in AKT stimulation, and may activate Ras either directly or via SRC. In this way, locally produced relaxin may regulate Sertoli cell function in a paracrine/autocrine manner.
Acknowledgments This work was supported by Fundac- a~ o de Amparo a Pesquisa do Estado de Sa~ o Paulo (FAPESP, Grant 2008/57239-7 to M.F.M.L.). Master (A.R.N.), Doctoral (M.T.P.) and postdoctoral (T.F.G.L.) fellowships were supported by FAPESP. Research fellowship (C.S.P. and M.F.M.L.) and doctoral fellowship (C.R.) were supported by Conselho Nacional do Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). We thank Espedita M. J. Silva Santos and Sheilla A. F. Fernandes for technical assistance, and Dr. G.H.M. Schoorlemmer for his valuable comments on this manuscript. References Bravo, R., Frank, R., Blundell, P.A., Macdonald, B., Macdonald, H., 1987. Cyclin/PCNA is the auxiliary protein of polymerase-d. Nature 326, 515–517. Cardoso, L.C., Nascimento, A.R., Royer, C., Porto, C.S., Lazari, M.F.M., 2010. Locally produced relaxin may affect testis and vas deferens function in rats. Reproduction 139, 185–196. Chambard, J.-C., Lefloch, R., Pouysse´gur, J., Lenormand, P., 2007. ERK implication in cell cycle regulation. Biochim. Biophys. Acta 1773, 1299–1310. Chang, F., Lee, J.T., Navolanic, P.M., Steelman, L.S., Shelton, J.G., Blalock, W.L., Franklin, R.A., Mccubrey, J.A., 2003. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 17, 590–603. Cheng, J.., Watkins, S.C., Walker, W.H., 2007. Testosterone activates mitogenactivated protein kinase via Src kinase and the epidermal growth factor receptor in Sertoli cells. Endocrinology 148, 2066–2074. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Chuang, C.C., Yang, R.S., Tsai, K.S., Ho, F.M., Liu, S.H., 2007. Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt,
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells Aline Rosa Nascimento, Maristela Taliari Pimenta, Thais F.G. Lucas, Carine Royer, Catarina Segreti Porto, Maria Fatima Magalhaes Lazari n ~ Paulo, Sao ~ Paulo, Brazil Section of Experimental Endocrinology, Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de Sao
a r t i c l e i n f o
abstract
Article history: Received 30 January 2012 Received in revised form 28 June 2012 Accepted 3 July 2012 Available online 20 July 2012
Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of Gi. AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24 h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was Gi- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to Gi and activation of both MEK/ERK1/2 and PI3K/AKT pathways. & 2012 Elsevier B.V. All rights reserved.
Keywords: Relaxin RXFP1 Sertoli cell Proliferation ERK1/2 PI3K/AKT
1. Introduction Relaxin is a member of the family of insulin-related peptides, first recognized for its important role during pregnancy and parturition, but now known to have several additional functions (Sherwood, 2004; Dschietzig et al., 2006). Relaxin is structurally similar to insulin, but binds to RXFP1 (relaxin family peptide receptor 1), a G-protein coupled receptor (GPCR) that belongs to the subfamily of leucine-rich repeat containing GPCRs (LGRs), which also includes the receptors for the glycoprotein hormones TSH, FSH and LH (Hsu et al., 2002). The role of relaxin in male reproduction is still unclear (Ivell et al., 2011). It was initially thought that relaxin was produced by the prostate and released to the seminal fluid to affect sperm motility (Sasaki et al., 2001; Sherwood, 2004). More recently, relaxin has been shown to influence sperm functionality (Ferlin et al., 2011; Miah et al., 2011). Although the major production of relaxin in the male reproductive tract occurs in the prostate,
n
Corresponding author. Fax: þ5511 5576 4448. E-mail addresses: [email protected] (A.R. Nascimento), [email protected] (M.T. Pimenta), [email protected] (T.F.G. Lucas), [email protected] (C. Royer), [email protected] (C.S. Porto), [email protected] (M.F.M. Lazari). 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.021
testes are also a source of relaxin (Cardoso et al., 2010; Gunnersen et al., 1995; Kohsaka et al., 2009). Studies with relaxin gene knockout animals (Rln / ) revealed that relaxin plays a role in the growth and development of the male reproductive tract (Samuel et al., 2003). The Rln / mice have smaller testis, decreased sperm maturation, and increased apoptosis (Samuel et al., 2003, 2005). Relaxin mRNA levels are higher in the testis of immature than adult rats, and Sertoli cells of immature rats represent an important source of relaxin mRNA (Cardoso et al., 2010). Relaxin induces proliferation of cultured rat Sertoli cells, suggesting an autocrine or paracrine role (Cardoso et al., 2010; Filonzi et al., 2007). Sertoli cells play an essential role in several stages during life. They are important for the expression of the male phenotype and to support spermatogenesis during puberty (Sharpe et al., 2003). The number of Sertoli cells will determine the extension of sperm production. There are several evidences that interplay between Ras/Raf/ MEK/ERK and PI3K/AKT pathways may be necessary for normal cell proliferation to occur (rev. Chambard et al., 2007), and both pathways are important for Sertoli cell function (Meroni et al., 2002; Cheng et al., 2007; Fix et al., 2004; Royer et al., 2012). GPCR agonists may promote cell proliferation and differentiation through the activation of these pathways (rev. Rozengurt, 2007).
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Ras/Raf/MEK/ERK and PI3K/AKT pathways may be activated by Gas, Gaq or bg subunits released from Gi or G0 (Yart et al., 2002; Stork and Schmitt, 2002). GPCRs may also activate the ERK1/2 and PI3K/AKT pathways through the transactivation of growth factor receptors (Schlessinger, 2000; Ohtsu et al., 2006; Rozengurt, 2007; Lucas et al., 2008), and through G protein-independent and arrestin-dependent mechanisms (Lefkowitz and Shenoy, 2005). The mechanism by which relaxin stimulates proliferation and differentiation is not yet clear. When overexpressed in HEK293T cells, RXFP1 couples with Gs, G0B, and Gi3, affecting cyclic AMP accumulation in a complex manner (Halls et al., 2006, 2007). Endogenous expression of RXFP1 allows a more selective coupling to G proteins, depending on the cellular context (rev. in Halls, 2012). This study investigated the pathways involved in the relaxin-stimulated proliferation of rat Sertoli cells, which endogenously express RXFP1 (Filonzi et al., 2007).
2. Material and methods 2.1. Primary cell culture The experimental procedures were approved by the Research Ethical Committee from UNIFESP-EPM (CEP0001/09). We used 15-day old male Wistar rats that were housed in the Animal Facility at the Instituto de Farmacologia e Biologia Molecular (INFAR), Escola Paulista de Medicina-Universidade Federal de Sa~ o Paulo (EPM-UNIFESP), and maintained on a 12 h light/12 h dark lighting schedule at 23 1C, with food and water freely available. The testes were removed and decapsulated, and Sertoli cells were isolated as previously described (Grima et al., 1995; Lucas et al., 2004, 2008). Cells were plated at a density of approximately 4 106 cells/100 mm dish (about 5 104 cells/ cm2), in phenol-red free Ham’s F12/Dulbecco’s Modified Eagle Medium (F12/DMEM 1:1, Gibco, Invitrogen, Grand Island, NY, USA) containing 0.02 g/l gentamicin (Sigma Chemical Co., St Louis, MO, USA), pH 7.2–7.4, and supplemented with 10 mg/ml insulin, 10 mg/ml transferrin, 10 ng/ml sodium selenite and 10 ng/ml epidermal growth factor (Sigma). Cells were grown in a humidified atmosphere of 5% CO2–95% air at 35 1C for 48 h, treated with 20 mM Tris–HCl, pH 7.4, to lyse residual germ cells (Galdieri et al., 1981), and allowed to grow for another 24 h. The culture was analyzed by morphological and immunocytochemical techniques (Lucas et al., 2008), confirming that the large majority of the population was Sertoli cells. Culture medium was replaced by another one without supplements 20 h before the experiments. At this stage, cells were 90–95% confluent and the viability, as determined by trypan blue exclusion, was higher than 90%. For proliferation assays, Sertoli cells were prepared as described above, plated at a low density, and at the day of experiments the confluence was 50–60%. 2.2. [Methyl-3H] thymidine incorporation assays Incorporation of [methyl-3H] thymidine into cell DNA was estimated as described by Guizzetti et al. (1996). Previous studies in our laboratory indicated that incorporation of [methyl-3H]thymidine (2 mCi/ml, specific activity 79.0 Ci/mmol, GE) in cultured Sertoli cells was time-dependent and linear from 2 to 10 h of incubation (Lucas et al., 2004). Therefore, primary Sertoli cell cultures on culture day 4 were initially incubated with 2 mCi/ml [methyl-3H]thymidine for 6 h at 35 1C. Afterwards, cells were incubated in the absence or presence of the MEK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2aminophenylthio]-butadiene (U0126, 20 mM, 30 min, Cell Signaling Technology, Danvers, MA), the PI3K inhibitor wortmannin (100 nM,
30 min, Sigma), the PKA inhibitor N-[2-[[3-(4-bromophenyl)-2propenyl]amino]ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 2 mM, 2 h, Sigma), the selective inhibitor of EGFR kinase, 4-(3chloroanilino)-6,7-dimethoxyquinazoline (AG1478, 1 mM, 15 min, Calbiochem., San Diego, CA, USA) or the PKC inhibitor GF 109203X (5 mM, 30 min, Sigma). Incubation was continued in the absence or presence of these inhibitors and in the absence or presence of recombinant human relaxin H2 (RLN; Phoenix Pharmaceuticals Co., Burlingame, CA, USA), 50 ng/ml, for 24 h at 35 1C. The reaction was stopped by cooling the cells at 0 1C. The medium was aspirated and the cells rinsed twice with ice-cold PBS and 5% trichloroacetic acid (Sigma). The cells were then solubilized with 0.5 N NaOH, collected with cotton-swabs, and transferred to 5 ml OptiPhase HiSafe-3 scintillation liquid (PerkinElmer Life Science Products, Boston, MA, USA). Bound radioactivity was determined in a scintillation b counter (LS 6000 IC, Beckman Colter Inc., Palo Alto, CA, USA). Results were expressed in relation to control, basal levels of [methyl-3H] thymidine incorporation (absence of relaxin and inhibitors) 2.3. Determination of cadherins 1 and 2 mRNA levels by real-time qRT-PCR 2.3.1. RNA extraction and cDNA synthesis Cells were incubated with 50 ng/ml of relaxin at 35 1C for 2 and 4 h. The reaction was stopped on ice, and cells were washed with cold sterile phosphate-buffered solution (PBS). Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the standard protocol (Chomczynski and Sacchi, 1987). RNA concentration was measured by spectrophotometry, and OD 260:280 nm ratios between 1.8 and 2.1 were obtained for all RNA samples. Ribosomal RNA integrity was checked by agarose gel electrophoresis, and a sharp and clear 2:1 ratio of ethidium bromide stained 28S:18S rRNA bands was observed for all samples. Two micrograms total RNA were used to synthesize the first strand cDNA at 50 1C with the Superscript III first strand synthesis Supermix (Invitrogen) and the oligo-dT primer supplied with this kit. 2.3.2. Quantitative PCR For the qPCR, we used the SYBR Green system (Applied Biosystems, Foster City, CA, USA). The following oligonucleotides were used to amplify fragments of cadherin 1 (Cdh1), cadherin 2 (Cdh2) and the endogenous control b-actin (Actb): Cdh1: 50 ACCGGCATCACCACAGAGACC-30 (forward) and 50 - CCGGGCAGTTGATGG GAGGG-30 (reverse); Cdh2: 50 -GCGGCCTTGCTTCAGGCATC30 (forward) and 50 -CTGGCCTTCGTGCACGTCCT-30 (reverse); Actb: 50 - GTAGCCATCCAGGCTGTG TT-30 (forward) and 50 -CCCTCATAGATGGGCACAGT-30 (reverse). The primers (Invitrogen) were designed with the Primer3 program (Rozen and Skaletsky, 2000) and spanned exon–exon boundaries whenever possible. Primers for target genes and beta-actin were designed to have approximately the same amplification efficiency (¼1). Controls without cDNA or without primers were included in each assay. Samples were run in a 7500 Real-Time PCR System (Applied Biosystems), using default conditions of amplification (50 1C for 2 min, 95 1C for 10 min and 40 cycles of 95 1C for 15 s and 60 1C for 1 min). The dissociation curves were obtained at the end to confirm specificity of the amplification. Each sample was run in triplicate. The average cycle threshold (CT) was determined with Applied Biosystems software. Data were analyzed by the comparative DDCT method (ABI PRISM User Bulletin #2, Applied Biosystems). The control values were always used as a calibrator in each experiment. Data are expressed as mean 7S.E.M. of the 2-DDCT from 3 different cDNAs (from three different cultures). At the end of the experiments,
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samples were run in 2% agarose gel electrophoresis to further confirm the absence of nonspecific amplification. The size of the expected products was compared to a DNA ladder (100 bp ladder, Invitrogen).
2.4. Western blot analysis On culture day 4, Sertoli cells were incubated in the absence (control) and presence of 25 or 50 ng/ml of relaxin, for the indicated times at 35 1C. In another series of experiments, before treatment with relaxin the cells were untreated or pretreated at 35 1C with the MEK1/2 inhibitor U0126 (20 mM, 30 min), the PI3K inhibitor wortmannin (100 nM, 30 min), the PKA inhibitor H89 (2 mM, 2 h), the Gi inhibitor pertussis toxin (PTX, 100 ng/ml, 16 h; Sigma), or the SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min; Calbiochemical, San Diego, CA, USA). At these concentrations, the inhibitors are highly selective, as previously reported in Sertoli and other cells (Cre´pieux et al., 2001; Lucas et al., 2008, 2010; Halls et al. 2006; Tai et al., 2009; Moore et al., 2007; Peters et al. 1999). Medium was removed, the cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, 10 mM Na4P207), as previously described (Lucas et al., 2008; Royer et al., 2012). Protein concentration was determined with a BioRad protein assay, using BSA as standard (Bio Rad Laboratories, Hercules, CA, USA).
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Total cellular proteins (30 mg/lane for AKT, and 60 mg/lane for ERK1/2 and PCNA) were incubated with sample buffer containing b-mercaptoethanol and subjected to SDS/PAGE. Proteins were electrotransferred onto PVDF membranes overnight, 20 V at 4 1C. Membranes were blocked in TBS containing 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk, pH 7.6, for 2 h at room temperature. Membranes were washed in TBS-T, and incubated overnight at 4 1C with primary antibody. To mark total and phosphorylated MAP kinase, we used rabbit polyclonal antibodies against rat p44/ p42 MAP kinase (#9102, Cell Signaling Technology), and phospho-p44/p42 MAP kinase (Thr202/Tyr204, #9101, Cell Signaling Technology) diluted 1:1000 and 1:2000, respectively, in blocking solution. Total and phosphorylated AKT were labeled with rabbit monoclonal antibody against a fragment in the C-terminal sequence of mouse AKT (pan-AKT, Cell Signaling Technology), and polyclonal antibody against phospho-Ser473 AKT (p-AKT, Ser473, Cell Signaling Technology), diluted in blocking solution, 1:2000 and 1:1000, respectively. Proliferating cell nuclear antigen (PCNA) was detected with a mouse monoclonal antibody (#2586, Cell Signaling Technology, 1:200). Membranes were next incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (Amersham Biosciences), and proteins were visualized with enhanced chemiluminescence reagent (ECL, GE Healthcare, Piscataway, NJ, USA). Apparent molecular weights were compared with molecular weight standards (New England Biolabs, Boston, MA, USA), and band intensities were quantified by densitometric analysis of linear-range immunoblots by using Epson Expression 1680 scanner (Epson America Inc., CA, USA) and
Fig. 1. Effects of relaxin on proliferation and differentiation of rat Sertoli cells. Effect of relaxin (A) and the inhibitors (B) on Sertoli cell proliferation. Cells were loaded with [methyl-3H]thymidine for 6 h at 35 1C, treated with U0126 (20 mM, 30 min), wortmannin (100 nM, 30 min), H89 (2 mM, 2 h), AG1478 (1 mM, 15 min), or GF109203X (5 mM, 30 min), and incubated in the absence (basal incorporation, control) or presence of relaxin (RLN, 50 ng/ml) for 24 h. Bound radioactivity was determined, and the results were expressed in relation to control (mean 7 S.E.M. of 3–4 independent experiments performed in triplicate). Basal incorporation of [methyl-3H] thymidine was 1440.7 7 164.4 dpm/well, and it was only affected by the PKC inhibitor GF 109203X (Fig. 1B). The asterisks indicate significant difference from control (ANOVA followed by Dunnett’s test, Po 0.05). (C) Western blot analysis of effect of relaxin (RLN, 50 ng/ml for 24 h) on the expression of the proliferating cell nuclear antigen, PCNA. The bar graph shows the densitometric analysis of the Western blots. Results were normalized to GAPDH expression and plotted in relation to control ( ¼ 1). Data are representative of 3 independent experiments. Error bars indicate S.E.M. * Po 0.05, Student’s t test. (D) Real-time PCR analysis of the gene expression of cadherins 1 (Cdh1) and 2 (Cdh2) after stimulation with RLN (50 ng/ml) for 2 and 4 h. Values were normalized using b-actin as endogenous control, and are expressed as relative values (means7 S.E.M.) of 3 independent experiments performed in triplicate. Differences between the three groups were not statistically significant (ANOVA).
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Quick Scan 2000 WIN software (Helena Laboratories, TX, USA). Results were normalized based on total ERK1/2, AKT or GAPDH expression and plotted (mean7S.E.M.) in relation to control, C (¼1). 2.5. Statistical analysis Data were expressed as mean 7S.E.M. Statistical analysis was performed with GraphPad Prism 5, version 5.01 (GraphPad Software Inc.). ANOVA was followed by Newman–Keuls for multiple comparisons, or Dunnett’s test for comparison with control. Student’s t-test was used for comparisons between two groups. P valueso0.05 were accepted as significant.
3. Results 3.1. Pathways involved in the relaxin-induced Sertoli cell proliferation Treatment of Sertoli cells with 50 ng/ml relaxin induced a 41% increase of cell proliferation (Fig. 1A), and almost doubled the expression of PCNA (Fig. 1C), which is an auxiliary protein of DNA polymerase d that serves as a marker for proliferation in most cells (Bravo et al., 1987). To determine the pathways involved in the relaxin-induced proliferation of rat Sertoli cells, we analyzed the effect of relaxin in the presence of specific inhibitors of several
pathways: the MEK1/2 inhibitor U0126, the PI3K inhibitor wortmannin, the PKA inhibitor H89, the inhibitor of EGFR activity AG1478, and the PKC inhibitor GF109203X. Relaxin-induced [3H]thymidine incorporation was inhibited by U0126, wortmannin, and H89, but not by GF109203X and AG1478. The inhibitors alone did not significantly affect the basal level of [3H]-thymidine incorporation, except GF109203X, which significantly increased Sertoli cell basal [3H]-thymidine incorporation (Fig. 1B). We also investigated whether relaxin affects the expression of cadherins 1 and 2, which are the constituents of the adherens junctions in testis, and markers of Sertoli cell differentiation (Siu and Cheng, 2004). Treatment of Sertoli cells with relaxin for 2 and 4 h failed to stimulate gene expression of cadherins 1 and 2 (Fig. 1D). 3.2. Relaxin stimulates a Gi-dependent MEK/ERK1/2 phosphorylation in rat Sertoli cells Treatment of primary culture of rat Sertoli cells with relaxin caused a time and concentration-dependent activation of ERK1 and ERK2 phosphorylation (Fig. 2A and B). The activation was rapid and transient, with a peak at 5 min and a complete return to basal control levels after 30 min (Fig. 2A). Based on these results, all subsequent experiments on relaxin-induced ERK1/2 phosphorylation were performed with 5 min incubations with 50 ng/ml relaxin. In addition, since relaxin induced similar activation of ERK1 and ERK2, only the combined phosphorylation of ERK1 and
Fig. 2. Relaxin activates a MEK-dependent ERK1/2 phosphorylation in a time and concentration-dependent fashion. (A) Western blot analysis of the time course of ERK1/2 phosphorylation by 50 ng/ml relaxin. (B) Concentration-dependent phosphorylation of ERK1/2 by 5 min incubation with relaxin. (C) Effect of the MEK1/2 inhibitor U0126 (20 mM, 30 min) on ERK1/2 phosphorylation induced by relaxin (50 ng/ml). (D) Effect of various inhibitors on basal ERK1/2 phosphorylation: MEK1/2 inhibitor U0126 (20 mM, 30 min), PI3K inhibitor wortmannin (100 nM, 30 min), PKA inhibitor H89 (2 mM, 2 h), Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h), and SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min). The bar graphs show the densitometric analysis of the Western blots. (A) and (B) Open bars: ERK1; solid bars: ERK2. (C) and (D) Combined expression of ERK1 and ERK2. Results were normalized to total ERK1/2 expression and plotted in relation to control ( ¼ 1). Error bars show S.E.M., * Po 0.05 (Student’s t test), n¼3–5 independent experiments. Different letters indicate differences among experimental groups (ANOVA and Newman–Keuls test, P o0.05).
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ERK2 is presented. Fig. 2C shows that relaxin-induced ERK1/2 phosphorylation was blocked by the MEK inhibitor U0126. Fig. 2D shows that U0126 and the several other inhibitors used in the subsequent experiments did not significantly affect the basal level of ERK1/2 phosphorylation. RXFP1 can couple with Gs, G0 and Gi (Halls et al., 2012), but Sertoli cells do not seem to express G0 (Paulssen et al., 1991). We investigated the involvement of Gs-cyclic AMP-PKA and Gi on the relaxin-induced ERK1/2 phosphorylation. Fig. 3A illustrates that pre-treatment of cells with the PKA inhibitor H89 reduced the effect of relaxin on ERK1/2-activation. Pretreatment of cells with 100 ng/ml pertussis toxin for 16 h completely blocked the effect of relaxin on ERK1/2 phosphorylation (Fig. 3B). The phosphorylation of ERK1/2 was inhibited by PP2, an inhibitor of the SRC-family of nonreceptor tyrosine kinases (Fig. 3C). In addition, activation of PI3K seems to contribute to relaxin-induced ERK1/2 activation, because pre-incubation with wortmannin blocked this effect (Fig. 3D).
3.3. Relaxin stimulates a Gi-dependent activation of PI3K/AKT pathway Considering that inhibition of PI3K inhibited relaxin-induced ERK1/2 phosphorylation, we investigated in more details the role of relaxin on the activation of PI3K/AKT pathway, measuring the ability of the hormone to induce phosphorylation of the
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AKT-Ser243 (Fig. 4). Fig. 4A shows that only wortmannin significantly affected the basal phosphorylation of AKT. A rapid increase in AKT phosphorylation was observed after 5 min incubation with relaxin, and this effect was completely blocked by pertussis toxin but not by H89 (Fig. 4B). Relaxin-induced phosphorylation of AKT was also completely blocked by the PI3K inhibitor wortmannin and by the MEK inhibitor U0126 (Fig. 4C). It is worth to mention that even after correcting the inhibitory effect of wortmannin on basal phosphorylation of AKT, this inhibitor was still able to completely block relaxin-induced AKT phosphorylation. The relaxin-induced activation of the PI3K/AKT pathway could still be detected after incubation with relaxin for 24 h (results not shown).
4. Discussion We have previously suggested that relaxin plays an autocrine/ paracrine role in testis (Cardoso et al., 2010; Filonzi et al., 2007). The use of pharmacological tools to inhibit specific signaling steps allowed us to suggest that both the MEK/ERK1/2 and the PI3K/ AKT pathway contribute to relaxin-induced Sertoli cell proliferation, and activation of a Gi protein and a SRC nonreceptor tyrosine kinase are the major upstream pathways to activate MEK1/2/ ERK1/2 and PI3K/AKT pathways (Fig. 5). The signaling mechanism underlying the mitogenic effect of relaxin seems to depend on the cell-type. In the prostate cancer
Fig. 3. Pathways involved in the relaxin-induced ERK1/2 phosphorylation. Western blot analysis of expression of phospho-ERK1/2 (top) and total ERK1/2 (bottom), in the absence (control) or presence of relaxin (RLN, 50 ng/ml for 5 min), after pre-incubation with (A) the PKA inhibitor H89 (2 mM, 2 h); (B) the Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h); (C) the SRC-tyrosine kinase inhibitor PP2 (5 nM, 30 min); and (D) PI3K inhibitor wortmannin (100 nM, 30 min). All lanes were run on the same gel, but irrelevant lanes were removed from the figure. The bar graphs show the combined expression of ERK1 and ERK2, quantified by densitometric analysis of the Western blots. Results were normalized to total ERK1/2 expression and plotted in relation to control ( ¼1). Error bars indicate S.E.M., n¼ 3–5 independent experiments. Different letters indicate differences among experimental groups (ANOVA and Newman–Keuls, Po 0.05).
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Fig. 4. Relaxin induces AKT (Ser473) phosphorylation in the Sertoli cells. (A) Densitometric analysis of the effect of the inhibitors on basal AKT phosphorylation: MEK1/2 inhibitor U0126 (20 mM, 30 min), PI3K inhibitor wortmannin (100 nM, 30 min), PKA inhibitor H89 (2 mM, 2 h), and Gi inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h). * different from control (ANOVA, followed by Dunnett’s test; P o0.05). (B) Effect of PTX and H89 on AKT (Ser473) phosphorylation induced by relaxin (RLN, 50 ng/ml) for 5 min, n¼2 independent experiments. (C) Effect of wortmannin and UO126 on AKT (Ser473) phosphorylation induced by relaxin (RLN, 50 ng/ml) for 5 min, n ¼3–4 independent experiments. The bar graphs show the densitometric analysis of the immunoblots. Results were normalized to total AKT expression and plotted in relation to control (¼ 1). Error bars indicate S.E.M. Different letters indicate differences between groups (ANOVA and Newman–Keuls test, P o 0.05).
Fig. 5. A model of the intracellular signaling pathways involved in the mitogenic effect of relaxin in Sertoli cells. Relaxin (RLN) binds to the RXFP1 receptor and induces the coupling of the receptor to the Gi protein. Released bg subunits activate a nonreceptor tyrosine kinase of the SRC-family, and the PI3K/AKT pathway, which may stimulate cell proliferation. SRC and PI3K activate the Ras/ Raf/MEK1/2/ERK1/2 pathway, which also contributes to cell proliferation. The site of action of inhibitors is indicated by the blunted arrows. In this model, wortmannin inhibits the PI3K-mediated activation of Ras/MEK/ERK1/2 pathway, and the MEK inhibitor U0126 may inhibit the PI3K/AKT pathway through the release of the activity of the phosphatase and tensin homolog (PTEN), an endogenous inhibitor of PI3K.
cell line LNCaP, relaxin is able to activate both Gas/cyclic AMP/ PKA and PI3K/AKT pathways to induce NFkB and co-translocation of AR and b-catenin to the nucleus (Vinall et al., 2011). Relaxininduced proliferation of cervical epithelial and stromal cells depends on stromal expression of the estrogen receptor ERa, which binds to estrogen response elements (Yao et al., 2010). In Sertoli cells of the rat, relaxin caused a rapid and transient increase of ERK1/2 phosphorylation, peaking after 5 min of
exposure to relaxin, and returning to basal levels after 30 min of exposure. Similar rapid relaxin-induced ERK1/2 activation was seen in human endometrial stromal cells, in the human acute monocytic leukemia cell line THP-1, in smooth muscle cells from the coronary artery, and in cells from human endometrial stroma (Zhang et al., 2002). However, this effect may be cell type dependent, because the relaxin-induced ERK1/2 activation in rat myofibroblasts involved both a rapid (2 min) and a lower but sustained (50 min) component (Mookerjee et al., 2009), and in endothelial cells of umbilical vein and in the cervical cancer human cells HELA, relaxin stimulated the ERK1/2 pathway only after a much longer period of stimulation (45–90 min) (Dschietzig et al., 2003). The PKA inhibitor H89 seemed to affect relaxin-induced Sertoli cell proliferation, and this inhibitor reduced relaxin-induced ERK1/2 phosphorylation, suggesting that the classical Gs/cyclic AMP/PKA pathway described for relaxin in other systems may contribute to the activation of the MEK/ERK1/2 pathway in Sertoli cells. Nevertheless, using an immunoenzymatic assay, we detected a robust accumulation of cyclic AMP in Sertoli cells induced by FSH or forskolin, but not by relaxin (unpublished results). We cannot exclude the possibility that relaxin stimulates a compartmentalized cyclic AMP production in Sertoli cells (Ostrom and Insel, 2004), which could not be easily detected by the immunoenzymatic assay in whole cell extract. ERK1/2 activation was sensitive to pertussis toxin, indicating a Gi-dependent mechanism, and to wortmannin, indicating a PI3Kdependent mechanism. It is well known that GPCRs can activate PI3K through the release of bg subunits from Gi and G0 (Curnock et al., 2002; Yart et al., 2002). Since Sertoli cells do not express G0 (Paulssen et al., 1991), bg subunits released from Gi are probably responsible for the relaxin-induced activation of PI3K. Activation of PI3K can activate the MEK/ERK1/2 pathway by directly phosphorylating Ras (Hawes et al., 1996) or SRC (Rameh et al., 1995). The activation of ERK1/2 by relaxin involved a SRC-dependent mechanism because it was inhibited by the SRC inhibitor PP2. Activation of SRC by relaxin could be mediated by bg subunits of Gi (Liebmann, 2011; Prenzel et al., 1999), or by upstream stimulation
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of PI3K, which generates PtdIns (3,4,5) P3 that is able to bind directly to the SH2 domain of SRC (Rameh et al., 1995). SRC mediates the phosphorylation of Ras, but may also phosphorylate Tyr-845 of the tyrosine kinase domain of EGFR and lead to ERK1/2 activation. However, the mitogenic effect of relaxin is not inhibited by AG1478, an inhibitor of the tyrosine kinase activity of EGFR. PI3K acts on phosphatidylinositols (PtdIns) from the plasma membrane to release (PtdIns)-3,4-P2 (PIP2) and PtdIns-3,4,5-P3 (PIP3). PIP3 activates the novel PKC types e, Z and d (sensitive to GF109203X) (Toker et al., 1994). Although PKC may phosphorylate Ras and activate the Raf/MEK/ERK cascade, the PKC inhibitor GF109203X did not inhibit relaxin-induced Sertoli cell proliferation. In fact, this inhibitor tended to increase cell proliferation, which could be caused by an inhibition of the expression of proteins involved in cell cycle by PKC (Livneh and Fishman, 1997). In conclusion, activation of PKC does not seem to contribute to the mitogenic effect of relaxin in Sertoli cells of the rat. Relaxin-induced AKT phosphorylation, like relaxin-induced ERK1/2 activation, involved the upstream activation of Gi, because it was blocked by pertussis toxin. The activation of the PI3K/AKT pathway by relaxin did not involve upstream activation of the Gs/ cyclic AMP/PKA pathway, because the PKA inhibitor H89 did not affect AKT phosphorylation. Basal phosphorylation of ERK1/2 and AKT was detected even in cells starved from medium supplementation for 20 h. This basal phosphorylation of AKT and ERK1/2 could be due to the action of growth factors, or other autocrine/paracrine factors produced by Sertoli cells, including relaxin. Nevertheless, only wortmannin significantly reduced basal AKT phosphorylation, and the inhibitors did not significantly affect the basal level of ERK1/2 phosphorylation or 3H-thymidine incorporation. Both ERK1/2 and PI3K pathways appear to stimulate Sertoli cell proliferation, and both pathways seem to modulate the activation of each other. This conclusion was based on the observations that AKT phosphorylation was inhibited by the MEK1/2 inhibitor U0126, and ERK1/2 phosphorylation was inhibited by the PI3K inhibitor wortmannin. Similarly, the adipogenic effect of the peroxisome proliferator-activated receptor g (PPARg) also involved activation of PI3K/AKT by ERK1/2 (Chuang et al., 2007). The activation of ERK1/2 pathway may lead to the phosphorylation and inhibition of the phosphatase and tensin homolog, PTEN (Marino et al., 2003), a phosphatase that dephosphorylates PIP3 and inhibits activation of AKT. The modulation of MEK/ERK1/2 activation by PI3K/AKT pathway may be explained not only by the PI3K-mediated activation of Ras, but also because AKT and the phosphoinositide-dependent kinase 1 (PDK1) may phosphorylate and activate the p21-activated kinase-2 (PAK-2), which phosphorylates Raf-1, and consequently leads to the activation of the MEK/ERK1/2 pathway (Siu et al., 2005). FSH and testosterone are important regulators of Sertoli cell function (Shupe et al., 2011). In primary culture of Sertoli cells isolated from 20-day old rats, FSH stimulates adenylyl cyclase and cyclic AMP production via activation of Gs, and PI3K pathway via activation of Gi (Meroni et al., 2002). The FSH-induced activation of ERK1/2 pathway depends on the dual coupling of the FSH receptor with Gs and Gi, in a PKA- and SRC-dependent manner (Cre´pieux et al., 2001). The activation of ERK1/2 by FSH is dependent on the developmental stage of Sertoli cells. FSH stimulates ERK1/2 phosphorylation and proliferation of Sertoli cells from immature rats, but inhibits the ERK pathway when the differentiation of Sertoli cells starts (Cre´pieux et al., 2001). Relaxin and FSH seem to use similar upstream signaling pathways to activate PI3K/AKT and MEK/ERK1/2 pathways, but whether relaxin also stimulates ERK1/2 phosphorylation in differentiating Sertoli cells still remains to be determined. It remains also to be determined whether relaxin plays a role in Sertoli cell
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differentiation. Although relaxin stimulates cadherin 2 expression in MCF7 breast cancer cells and uterus (Sacchi et al., 1994; Ryan et al., 2001), relaxin did not affect gene expression of cadherins 1 and 2 in Sertoli cells. It is well known that the Ras/Raf/MEK/ERK and PI3K/AKT signaling cascades affect cell cycle regulation and cell proliferation. Several ERK targets that affect cell proliferation have been identified, and full induction of some cell cycle players sometimes requires activation of both the MEK/ERK and the PI3K/AKT pathways (rev. in Chambard et al., 2007). We still need to explore in more detail the pathways that follow the relaxin-mediated activation of PI3K/AKT and MEK/ERK1/2 pathways, but activation of transcription factors such as Elk-1 and c-Fos, or a direct effect of PI3K on the expression of proteins that affect cell cycle may be involved (Chang et al., 2003). Relaxin increased proliferation and the expression of PCNA in cultured leiomyoma cells but not in normal myometrial cells (Suzuki et al., 2012). Treatment of immature Sertoli cells with relaxin for 24 h also increased expression of PCNA, which is essential for the S-phase of the cell cycle and represents an important index of cell proliferation. Phosphorylation of the cell cycle inhibitor p21 by PI3K/AKT pathway releases PCNA from the complex p21/cdk1/cdk2/cyclins, allowing its association with DNA polymerase d and the transcription complex AP-1, inducing DNA synthesis and reducing ¨ inhibition of Cdk2 by p21 (Rossig et al., 2001). In conclusion, relaxin activates the PI3K/AKT and MEK-ERK1/2 pathways to induce proliferation of Sertoli cells. We propose the following sequence of events for the mitogenic effect of relaxin (Fig. 5): (1) Relaxin binds to RXFP1 and induces coupling to Gi; (2) released bg subunits activate PI3K and SRC; (3) PI3K activation results in AKT stimulation, and may activate Ras either directly or via SRC. In this way, locally produced relaxin may regulate Sertoli cell function in a paracrine/autocrine manner.
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