Identification of Smad Response Elements in the Promoter of Goldfish FSHβ Gene and Evidence for Their Mediation of Activin and GnRH Stimulation of FSHβ Expression

Man-Tat Lau,Wei Ge,Sze-Wah Lin

doi:10.3389/fendo.2012.00047

Abstract

As an essential hormone regulating gonads in vertebrates, the biosynthesis and secretion of follicle-stimulating hormone (FSH) is controlled by a variety of endocrine and paracrine factors in both mammalian and non-mammalian vertebrates. Activin was initially discovered in the ovary for its specific stimulation of FSH secretion by the pituitary cells. Our earlier studies in fish have shown that activin stimulates FSHβ but suppresses LHβ expression in both the goldfish and zebrafish. Further experiments showed that the regulation of FSHβ in fish occurred at the promoter level involving Smads, in particular Smad3. To further understand the mechanisms by which activin/Smad regulates FSHβ transcription, the present study was undertaken to analyze the promoter of goldfish FSHβ gene (fshb) with the aim to identify potential cis-regulatory elements responsible for activin/Smad stimulation. Both serial deletion and site-directed mutagenesis were used, and the promoter activity was tested in the LβT-2 cells, a murine gonadotroph cell line. The reporter constructs of goldfish FSHβ promoter-SEAP (secreted alkaline phosphatase) were co-transfected with an expression plasmid for Smads (2 or 3) followed by measurement of SEAP activity in the medium. Two putative Smad responsive elements were identified in the promoter at distal and proximal regions, respectively. The distal site contained a consensus Smad binding element (AGAC, −1675/−1672) whereas the proximal site (GACCTTGA, −212/−205) was identical to an SF-1 binding site reported in humans, which was preceded by a sequence (AACACTGA) highly conserved between fish and mammals. The proximal site also seemed to be involved in mediating stimulation of FSHβ expression by gonadotropin-releasing hormone and its potential interaction with activin. In conclusion, we have identified two potential cis-regulatory elements in the promoter of goldfish FSHβ that are responsible for activin-induced expression of the gene. Since activin stimulation of FSHβ expression is functionally conserved in fish and mammals, our findings contribute to the understanding of the fundamental mechanisms of this regulation across vertebrates.

Highlights

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal development and function in vertebrates including teleosts
The biosynthesis of follicle-stimulating hormone (FSH) in the pituitary is primarily determined by the expression of its β subunit (Shupnik, 1996), which is subject to regulation by a variety of neuroendocrine, endocrine, and paracrine factors including gonadotropin-releasing hormone (GnRH) from the hypothalamus (Ferris and Shupnik, 2006), sex steroids from the gonads (Glidewell-Kenney et al, 2008), and local factors in the pituitary, activin and its binding protein follistatin (Kawakami et al, 2002; Bilezikjian et al, 2004)
PROMOTER ANALYSIS FOR POTENTIAL Smad REGULATORY ELEMENT(S) IN GOLDFISH fshb GENE We previously showed that goldfish fshb promoter strongly responded to activin in the LβT-2 cells (Ge et al, 2003), and co-transfection with Smad expression vectors, especially Smad3, dramatically enhanced the expression level of the reporter, suggesting the presence of Smad regulatory elements in the promoter (Lau and Ge, 2005)

Summary

Introduction

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal development and function in vertebrates including teleosts. Subsequent studies have provided evidence that activin is produced locally in the pituitary together with its neutralizing binding protein follistatin in both mammals and fish (Meunier et al, 1988; Roberts et al, 1989; Lau and Ge, 2005; Lin and Ge, 2009) and the pituitary-derived activin plays an important role in controlling FSH biosynthesis and secretion in a paracrine manner (Bilezikjian et al, 2004; Gregory and Kaiser, 2004). Activin stimulates fshb mRNA expression in the goldfish (Yam et al, 1999), tilapia (Yaron et al, 2001), zebrafish (Lin and Ge, 2009), and European eel

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