Pascale Crépieux

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

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

Mapping the follicle-stimulating hormone-induced signaling networks

Follicle-stimulating hormone (FSH) is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signaling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signaling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signaling network. We discuss the multiple facets of FSH-induced signaling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signaling network represent a first step toward gaining a system-level comprehension of this hormone’s actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertility and non-steroidal contraception.

Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT1AR, and HEK293 cells expressing other 7TMRs.

Preferential β-arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189 V mutation.

The A189 V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189 V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through β-arrestins, independently of the canonical cAMP/PKA pathway. Interesting, both the A189 V and wild-type (Wt) FSHRs selectively activated cAMP-independent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189 V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSHβ are more deleterious to human fertility than the FSHR A189 V mutation which preserves parts of receptor signalling repertoire.

Novel pathways in gonadotropin receptor signaling and biased agonism

Gonadotropins play a central role in the control of male and female reproduction. Selective agonists and antagonists of gonadotropin receptors would be of great interest for the treatment of infertility or as non steroidal contraceptive. However, to date, only native hormones are being used in assisted reproduction technologies as there is no pharmacological agent available to manipulate gonadotropin receptors. Over the last decade, there has been a growing perception of the complexity associated with gonadotropin receptors’ cellular signaling. It is now clear that the Gs/cAMP/PKA pathway is not the sole mechanism that must be taken into account in order to understand these hormones’ biological actions. In parallel, consistent with the emerging paradigm of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. Small molecule ligands, modulating antibodies interacting with the hormones and glycosylation variants of the native glycoproteins have all demonstrated their potential to trigger such selective signaling. Altogether, the available data and emerging concepts give rise to intriguing opportunities towards a more efficient control of reproductive function and associated disorders.

Partially Deglycosylated Equine LH Preferentially Activates β-Arrestin-Dependent Signaling at the Follicle-Stimulating Hormone Receptor

Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.

Biased signalling in follicle stimulating hormone action

Follicle-stimulating hormone (FSH) plays a crucial role in the control of reproduction by specifically binding to and activating a membrane receptor (FSHR) that belongs to the G protein-coupled receptor (GPCR) family. Similar to all GPCRs, FSHR activation mechanisms have generally been viewed as a two-state process connecting a unique FSH-bound active receptor to the Gs/cAMP pathway. Over the last decade, paralleling the breakthroughs that were made in the GPCR field, our understanding of FSH actions at the molecular level has dramatically changed. There are numerous facts indicating that the active FSHR is connected to a complex signalling network rather than the sole Gs/cAMP pathway. Consistently, the FSHR probably exists in equilibrium between multiple conformers, a subset of them being stabilized upon ligand binding. Importantly, the nature of the stabilized conformers of the receptor directly depends on the chemical structure of the ligand bound. This implies that it is possible to selectively control the intracellular signalling pathways activated by using biased ligands. Such biased ligands can be of different nature: small chemical molecules, glycosylation variants of the hormone or antibody/hormone complexes. Likewise, mutations or polymorphisms affecting the FSHR can also lead to stabilization of preferential conformers, hence to selective modulation of signalling pathways. These emerging notions offer a new conceptual framework that could potentially lead to the development of more specific drugs while also improving the way FSHR mutants/variants are functionally characterized.

G Protein-Coupled Receptor Kinases and Beta Arrestins Are Relocalized and Attenuate Cyclic 3′,5′-Adenosine Monophosphate Response to Follicle-Stimulating Hormone in Rat Primary Sertoli Cells

Abstract The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and β-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/β-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and β-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and β-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and β-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/β-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells.

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.

FSH-stimulated PTEN activity accounts for the lack of FSH mitogenic effect in prepubertal rat Sertoli cells

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.