LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site

Abstract

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to −715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to −402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the −715/−500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at −698/−688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at −698/−688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

Follicular fluid exosomes: Important modulator in proliferation and steroid synthesis of porcine granulosa cells

“…2 Proliferation and steroid synthesis of GCs are regulated by many factors, including IGF, BDNF, LH, and FSH. [3][4][5][6] The follicular fluid exosomes are new molecules in follicular fluid, 7 exploring the exosomes will help us to understand the new mechanism of follicular development.…”

“…The maturation or atresia of the follicle depends on the granulosa cells (GCs) state, particularly, GCs proliferation, and steroid synthesis are important in follicular development 2 . Proliferation and steroid synthesis of GCs are regulated by many factors, including IGF, BDNF, LH, and FSH 3‐6 . The follicular fluid exosomes are new molecules in follicular fluid, 7 exploring the exosomes will help us to understand the new mechanism of follicular development.…”

Preventing Growth Stagnation and Premature LH Surge Are the Keys to Obtaining a Viable Embryo in Monofollicular IVF Cycles: A Retrospective Cohort Study

“…After LH binds to its receptor LHCGR in granulosa cells, it simultaneously induces the activation of multiple intracellular signaling cascades, including the classical cAMP/PKA (Cyclic adenosine 3′,5′-monophosphate/protein kinase A) pathway [ 25 ], resulting in the phosphorylation of ERK1/2 (extracellular-regulated kinase) and CREB (cAMP-responsive element binding protein) and the transcription of steroidogenic genes, such as StAR (steroidogenic acute regulatory protein), P450scc (cytochrome P450 side chain cleavage) and aromatase, leading to an elevation of estrogen synthesis [ 26 , 27 , 28 ]. Apart from the steroidogenic effect, LH also promotes granulosa cell proliferation [ 29 ] and mediates anti-apoptotic effects [ 30 ] via the PI3K/AKT (phosphatidylinositol 3-kinases/protein kinase B) pathway by the activation of STARD1, CCND2 and XIAP gene expression [ 31 ]. It is not surprising that reduced LH activities would lead to apoptosis of ovarian granulosa cells [ 32 ].…”

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours

“…In our previous studies, we did not observe CREB-associated proteins forming complexes with the proximal Nppc promoter in vitro, but did identify binding of both Sp1 and Sp3 [ 11 ]. Sp1, in particular, is known to be cAMP responsive in gonadal tissue, where it mediates the LH response on Klf4 expression [ 65 ], and in human granulosa cells where FSH regulated Sp1-dependent IRS2 expression [ 66 ]. In our current study, of the three natriuretic peptide receptors, only Npr1 expression was significantly altered by forskolin, after 24 h treatment.…”