Gene analysis of major signaling pathways regulated by gonadotropins in human ovarian granulosa tumor cells (KGN)†

Abstract

The female reproductive function largely depends on timing and coordination between follicle-stimulating hormone (FSH) and luteinizing hormone. Even though it was suggested that these hormones act on granulosa cells via shared signaling pathways, mainly protein kinases A, B, and C (PKA, PKB, and PKC), there is still very little information available on how these signaling pathways are regulated by each hormone to provide such differences in gene expression throughout folliculogenesis. To obtain a global picture of the principal upstream factors involved in PKA, PKB, and PKC signaling in granulosa cells, human granulosa-like tumor cells (KGN) were treated with FSH or specific activators (forskolin, SC79, and phorbol 12-myristate 13-acetate) for each pathway to analyze gene expression with RNA-seq technology. Normalization and cutoffs (FC 1.5, P ≤ 0.05) revealed 3864 differentially expressed genes between treatments. Analysis of major upstream regulators showed that PKA is a master kinase of early cell differentiation as its activation resulted in the gene expression profile that accompanies granulosa cell differentiation. Our data also revealed that the activation of PKC in granulosa cells is also a strong differentiation signal that could control “advanced” differentiation in granulosa cells and the inflammatory cascade that occurs in the dominant follicle. According to our results, PKB activation provides support for PKA-stimulated gene expression and is also involved in granulosa cell survival throughout follicular development. Taken together, our results provide new information on PKA, PKB, and PKC signaling pathways and their roles in stimulating a follicle at the crossroad between maturation/ovulation and atresia.

Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN)

“…The ability of melatonin to inhibit granulosa cell proliferation and angiogenesis could be part of the mechanism driving granulosa cell early differentiation by highlighting beta-catenin (CTNNB1) expression in our results. The CTNNB1 protein is an estrogen synthesis regulator and a key factor of the Wnt signaling pathway [ 43 ].…”

“…The effects of melatonin are mediated through specific receptors, which activate numerous signaling pathways, such as the PKA pathway, in several tissues [ 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 ]. Therefore, forskolin was used to identify the effects of melatonin linked to PKA activation.…”

Gene cascade analysis in human granulosa tumor cells (KGN) following exposure to high levels of free fatty acids and insulin

“…The actions of both gonadotrophins, FSH and LH, are mediated via PKA and AKT signaling. PKA is a master kinase for granulosa cell differentiation and proliferation while AKT acts as a permissive pathway for PKA signaling and regulates cell survival [ 27 , 63 – 65 ]. Consistent with our results, obesity in women was associated with lower levels of estradiol [ 46 , 66 ].…”

“…As described in a previous study, mRNA was isolated from total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module (E7490S; NEB) and was fragmented to a mean size of 200 nt. Fragments were reverse transcribed to generate double-stranded cDNA and converted to a paired-end library using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530S; NEB) according to the manufacturer’s instructions and using Agencourt AMPure XP beads and NEBNext Mutliplex Oligos for Illumina (set1, E7335S; NEB) [ 27 ]. The libraries were sent to the Génome Québec Innovation Centre at McGill University (Montreal, Quebec, Canada) for quality control tests and then pooled at equimolar concentrations and sequenced on Illumina HiSeq4000 in paired-end mode with 100 base pair reads (PE100) to a depth between 53–69 million reads.…”

“…The sequence data produced in this study were submitted to NCBI GEO under accession number GSE161914. As described in a previous study, normalized data were sorted for significance and then filtered according to the Transcript Support Level (TSL) used by the Ensembl gene annotation system [ 27 ]. This annotation system is a method to highlight the well supported and poorly supported transcript models for users.…”