A Proteomic Comparison of Immature and Mature Mouse Gonadotrophs Reveals Novel Differentially Expressed Nuclear Proteins that Regulate Gonadotropin Gene Transcription and RNA Splicing1

Abstract

The αT3-1 and LβT2 gonadotroph cell lines contain all the known factors required for expression of gonadotropin genes, yet only the LβT2 cells express the β-subunits. We hypothesized that comparison of their nuclear proteomes would reveal novel proteins and/or modifications that regulate expression of these genes. We identified nine proteins with different expression profiles in the two cell lines, of which several were chosen for further functional studies. Of those found at higher levels in αT3-1 nuclei, 1110005A23RIK was found associated with the Fshb gene promoter and repressed its expression; TAGLN3 over-expression reduced transcript levels of Fshb, and its knockdown elevated Lhb and Cga transcript levels, indicating an on-going repressive effect on these more highly expressed genes, possibly through altering levels of phosphorylated MAPK; HNRNPA2B1 repressed splicing of the Fshb primary transcript, which it binds in the first intron. Proteins at higher levels in LβT2 nuclei included prohibitin, whose over-expression reduced promoter activity of all three gonadotropin subunits and appeared to mediate the differential effect of GnRH on proliferation of the two cell lines; its knockdown also altered cell morphology. Two other splicing factors were also found at higher levels in LβT2 nuclei: the knockdown of PRPF19 or EIF4A3 decreased splicing of Lhb, or of both β-subunit transcripts, respectively. The levels of Eif4a3 mRNA were increased by activin and both factors increased Fshb splicing. This study has revealed a number of novel factors that alter gonadotropin expression and gonadotroph function, and likely mediate or moderate effects of the regulatory hormones.

Lipid Raft- and Protein Kinase C-mediated Synergism between Glucocorticoid- and Gonadotropin-releasing Hormone Signaling Results in Decreased Cell Proliferation

“…Our finding that GnRH alone decreases cell proliferation in L␤T2 cells is consistent with a previous report (64). The insensitivity of this effect to both Flot-1 and SGK-1 knockdown taken together with the gene expression and ChIP results suggests that the mechanism of repression of cell proliferation by GnRH does not involve lipid raft-associated GR or SGK-1 expression.…”

Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

“…Luciferase assay constructs were created by ligating 1,300 bp of the proximal murine LH␤ gene promoter or 600 bp of the proximal murine FSH␤ gene promoter into pGL2 Basic (Promega), as reported previously (11,39). The expression vectors for Pitx1, Egr-1, and SF-1 were gifts from J. Drouin (Montreal, Canada), J. Milbrandt (St. Louis, MO), and K. Parker (Durham, NC), respectively.…”

“…The protein-bound DNA precipitated from the ChIP assay and input DNA were purified with phenol-chloroform and dissolved in 25 l H 2 O. Precipitated and input DNA served as templates for promoterspecific PCR. Primers used to amplify the FSH␤ and LH␤ gene-proximal promoters were as described previously (11).…”

Gonadotropin gene transcription is activated by menin-mediated effects on the chromatin

“…Nucleic and cytoplasmic proteins were isolated (as in [33]) using the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL). The proteins were resolved by electrophoresis on 10 to 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and were subsequently transferred on to an Immuno-Blot poly-vinylidene difluoride membrane (Bio-Rad, Hercules, CA) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS and 20% methanol).…”