The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation

Constitutive activation of the Ras/Raf/extracellular signal-regulated kinase (ERK) 3 signaling pathway has long been associated with cancers.Recently, activating point mutations in B-RAF were detected in a large scale screen for genes mutated in human cancer (1). About 80% of the B-Raf mutations seen in human cancers have the valine at position 600 in the activation segment substituted by a glutamic acid, which corresponds to the hot spot transversion T1799A mutation. Reconstitution experiments have shown that this mutation increases the activity of ectopically expressed B-Raf kinase and induces cell transformation (1, 2), whereas siRNA-mediated suppression of V600E B-Raf abolishes cell transformation (3). This gain of function mutation in B-RAF ( V600E BRaf) is largely responsible for the constitutive activation of ERK1/2 and is therefore for the acquisition of self-sufficiency in the growth signal in human cutaneous melanoma cells (1, 3, 4). Activating mutations in B-RAF and RAS are associated with about 60 and 20% of cutaneous melanoma, respectively (1, 5, 6). However, about 20% of cutaneous melanoma have no mutations in either B-RAF or RAS. This suggests that other molecular mechanisms activate the ERK signaling pathway. Consistent with this, the overexpression of B-Raf without gene amplification has been shown to be one of the mechanisms by which the constitutive activation of ERK1/2 stimulates cell growth in cutaneous melanoma cell lines expressing WT B-Raf and WT N-Ras (7). However, in melanoma cell lines that express normal levels of WT B-Raf, no constitutive activation of ERK1/2 and no implication of ERK1/2 in cell proliferation have been seen (8). The precise effect of WT B-Raf overexpression on the constitutive activation of ERK1/2 is still unknown, since reconstitution experiments showed that ectopically overexpressed WT B-Raf did not activate ERK1/2 in COS cells (9). During malignant transformation, cutaneous melanoma cells also acquire the ability to overexpress growth factor receptors and to express and secrete growth factors that together activate the ERK1/2 signaling pathway. It is therefore likely that autocrine growth factor activation loops are also involved in the acquisition of autonomous growth via the constitutive activation of ERK1/2 in cutaneous melanoma (6).Unlike cutaneous melanoma, which has been extensively studied, little is known about the molecular pathogenesis of ocular (uveal and conjunctival) melanoma. Uveal melanoma is the most common primary ocular neoplasm in adult humans in developed countries. Although cutaneous and uveal melanomas share similar histological features, mutations in the genes encoding the major signaling pathway proteins that control cell proliferation, including those affected in cutaneous melanoma, are infrequent in uveal melanoma (10 -17). This is consistent with the differences in the epidemiological and cytogenetic aspects of cutaneous and uveal melanomas. We recently demonstrated a large increase in the activity of endogenous B-Raf kinase and a cons...

Section: Discussionmentioning

confidence: 99%

Extracellular Signal-regulated Kinase-dependent Proliferation Is Mediated through the Protein Kinase A/B-Raf Pathway in Human Uveal Melanoma Cells

Calipel

Mouriaux

Glotin

et al. 2006

“…In general, FSH mainly signals via cAMP-dependent PKA activation, although it also activates the MEK/ERK cascade. This occurs either in parallel via a differentially spliced receptor (18) or downstream of cAMP (20). MEK/ERK-dependent FSH effects are readily abrogated by the specific MEK inhibitor PD98059 in porcine granulosa cells (18).…”

Section: Discussionmentioning

confidence: 99%

“…Notably, FSH does not act solely via cAMP, since it also stimulates mitogen-activated protein kinase pathways via an alternatively spliced FSH receptor in porcine granulosa cells (18). Furthermore, FSH can activate mitogen-activated protein kinase pathways downstream of PKA in mammalian granulosa cells (19) as well as Sertoli cells (20,21). There is increasing evidence suggesting that activins are not only endocrine regulators of FSH availability but also potent effectors of autocrine or paracrine intraovarian signaling.…”

mentioning

confidence: 99%

Activin A Signaling Induces Smad2, but Not Smad3, Requiring Protein Kinase A Activity in Granulosa Cells from the Avian Ovary

Schmierer

Schuster

Shkumatava

et al. 2003

Activin A signaling is an important regulator of ovarian granulosa cell function. The cytosolic signal transducer Smad2 is most highly expressed in chicken granulosa cells (cGC) of preovulatory follicles. Moreover, Smad2 shows predominant nuclear localization in freshly isolated cGC, indicating active Smad signaling in vivo. Primary cGC cultured in vitro require activin A to sustain high Smad2 levels, which otherwise drop dramatically in the absence of activin A. This activin A-dependent Smad2 expression is abrogated by protein kinase A (PKA) inhibitors, suggesting a role for PKA in activin signaling. In the absence of activin A, strong PKA activators such as follicle-stimulating hormone (FSH) and 8-bromo-cyclic AMP fail to elicit Smad2 induction. However, FSH and 8-bromo-cyclic AMP boost activin A-dependent Smad2 up-regulation, giving rise to Smad2 levels similar to expression in vivo levels. Interestingly, the effect is specific for Smad2, since expression of the structurally and functionally closely related Smad3 remains entirely unaffected. Hence, activin A induces Smad2, but not Smad3, to high levels requiring PKA activation. Since Smad2 and Smad3 target distinct yet overlapping sets of TGF-␤/activin-responsive genes, the selective Smad2 induction by FSH/activin A could allow FSH to efficiently modulate the transcriptional readout of activin A signaling in avian granulosa cells.

“…On this note, we anticipated that endogenous protein-tyrosine phosphatase inhibitors could protect samples from unwanted activities of proteintyrosine phosphatase; however, a complete blockade of protein-tyrosine phosphatase could shift the base line of the intrinsic kinase activity, but plausibly not the trend of activation. Furthermore, EGF (a well known receptor protein-tyrosine kinase), which was present in the Sertoli cell cultures at 2.5 ng/ml as a growth factor in our experiments as described previously (44,45), has recently been shown to stimulate Sertoli cell ERK activity at 100 ng/ml (46,47). As such, additional controls were included in Sertoli-germ cell co-cultures in which EGF was omitted in the nutrient mixture F-12/DMEM.…”

Section: Methodsmentioning

confidence: 99%

Sertoli-Germ Cell Anchoring Junction Dynamics in the Testis Are Regulated by an Interplay of Lipid and Protein Kinases

Siu

Wong

Lee

et al. 2005

173

196

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as ␤1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin⅐laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-␤1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the ␤1-integrin/PI3K/PKB/ERK signaling pathway.In the seminiferous epithelium of the rat testis, Sertoli-germ cell adhesion is maintained by cell-cell actin-based adherens junctions (AJs) 1 and intermediate filament-based desmosomelike junctions (for reviews, see Refs. 1-3). The best studied testis-specific AJ type is ectoplasmic specialization (ES). The ES is confined between Sertoli cells (known as the basal ES) at the site of the blood-testis barrier (BTB) as well as between Sertoli cells and spermatids (known as the apical ES) in the adluminal compartment of the epithelium (for reviews, see Refs. 3 and 4). Most of the studies on cell adhesion function in the testis in the past 2 decades have focused on the apical ES because the biochemical composition of the desmosome-like junction remains largely unexplored in the testis (for reviews, see Refs. 1, 3, and 5). The apical ES is an important anchoring junction device t...