Salt Inducible Kinases are Negative Regulators of Follicle Stimulating Hormone in Ovarian Granulosa Cells

Infertility is a significant and growing problem that affects about 12% of couples in the US. The strongest reason for this issue is a combination of the inevitable limitation of aging on fertility and the social tendency for couples to wait longer until they have children. Out of the about seven million women affected by infertility, 40% of them experience problems due to a dysfunction in ovulation. Ovulation is the culmination of a long process of follicle growth and maturation, termed folliculogenesis, in the ovaries. The main hormone responsible for driving folliculogenesis is the pituitary gonadotropin follicle-stimulating hormone (FSH). Local factors secreted in the follicle also play an important role for proper follicle maturation. One such factor is insulin-like growth factor (IGF). We have shown that the activity of the IGF1 receptor (IGF1R) is necessary and required for proper follicular response to FSH. Accordingly, high follicular levels of IGF in patients seeking assisted reproductive technologies, such as in vitro fertilization (IVF), are associated with a better response to FSH and better embryo quality. The exclusive target of FSH in the follicle are the granulosa cells (GCs) that surround the oocyte. FSH, through cAMP signaling, causes GC differentiation leading to their production of aromatase, an enzyme that converts androgens to estradiol. GC differentiation is required for follicle maturation, without which the follicle fails to develop and ovulate. A lack of IGF1R activity in GCs renders them unable to respond to FSH, and they fail to produce aromatase. Based on this, we postulated that IGF augments GC differentiation by inhibiting a negative regulator of FSH. Previous work in the lab has shown that IGF1R and FSHR activity converge on the activation of AKT, however, the downstream signals linking AKT to GC-differentiation are unknown. We hypothesized that the salt inducible kinases (SIKs), which are known to be inhibited by AKT and are known inhibitors of cAMP signaling, limit the response of the GCs to FSH. Here, I show that SIKs inhibition in GCs augments their response to FSH and production of aromatase in vitro and in vivo. I also investigate the placement of SIKs activity within the FSHR/IGF1R pathway. By understanding the signaling components that control follicle maturation, we can contribute to alleviating the emotionally and financially taxing problem of infertility

Image_3_Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.jpeg

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.</p

Image_1_Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.jpeg

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.</p

Image_2_Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.jpeg

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.</p