Abstract
Luteinizing hormone (LH) is synthesized and secreted throughout the reproductive cycle from gonadotrope cells in the anterior pituitary, and is required for steroidogenesis and ovulation. LH contains an α-subunit common with FSH, and a unique LHβ subunit that defines biological activity. Basal LHβ transcription is low and stimulated by hypothalamic GnRH, which induces synthesis of early growth response protein-1 (Egr1), and stimulates binding of transcription factors Egr1 and steroidogenic factor-1 (SF1) on the promoter. WT1 (Wilms tumor protein1) is a zinc finger transcription factor with an essential role in urogenital system development, and which regulates several reproductive genes via interactions with SF1 or binding to GC-rich elements such as Egr1 binding sites. We investigated a potential role for WT1 in LHβ transcription in clonal mouse gonadotrope LβT2 cells. WT1 was present in LβT2 and mouse pituitary cells, and protein bound to the endogenous LHβ promoter. Interestingly, mRNAs for WT1(+KTS), which contains a three amino-acid insertion between the 3rd and 4th zinc fingers, and the WT1 (-KTS) variant were both expressed at significant levels. WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated. Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms. Overexpression of WT1 showed that WT1(-KTS) enhanced LHβ promoter GnRH stimulation 2-to-3-fold and required the 3’Egr1 site, but WT1(+KTS) repressed both basal and GnRH-stimulated LHβ promoter activity by approximately 70%. Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.
Highlights
Gonadotropin hormones secreted from the anterior pituitary control female reproduction, and Luteinizing Hormone (LH) is necessary for ovulation and steroidogenesis [1, 2]
WT1 protein is expressed under basal conditions in LβT2 cells, whereas early growth response protein-1 (Egr1), a zinc-finger transcription factor proven to associate with the LHβ promoter, is not expressed at detectable levels under the same conditions (Fig. 1B)
To determine if WT1 could bind to the endogenous LHβ promoter, chromatin immunoprecipitation assays were performed in untreated LβT2 cells
Summary
Gonadotropin hormones secreted from the anterior pituitary control female reproduction, and Luteinizing Hormone (LH) is necessary for ovulation and steroidogenesis [1, 2]. In response to GnRH, co-ordinated binding of transcription factors occurs on the LHβ promoter [10, 15] These proteins in turn may associate with additional stimulatory and suppressive regulatory proteins, including SNURF [15], SRC-1 [16] and DAX-1 [17, 18] that influence the response of reproductive genes to hormonal and physiological challenges. WT1 binds directly to DNA at GC-rich motifs similar to those for Egr or Sp1 [22, 23] In spite of these intriguing associations with the transcription factors involved in LHβ gene transcription, the potential role of WT1 in LHβ gene transcription has not previously been examined. The +KTS variant represses both basal and GnRH stimulated LHβ transcription whereas the –KTS variant activates GnRH-stimulated LHβ transcription
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