Characterization of sea bass FSHβ 5′ flanking region: transcriptional control by 17β-estradiol

Abstract

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHβ 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHβ gene. Analysis of the ~3.5 kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5 kb of the sbFSHβ 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LβT2) cell lines. The sbFSHβ 5' FR was efficiently expressed under basal conditions in LβT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHβ transcriptional activity, in vitro treatments with 17β-estradiol were carried out on primary cultures of pituitary cells and LβT2 cells transiently expressing luciferase under the control of sbFSHβ 5' FR. Overall, these results demonstrate that 17β-estradiol inhibits sbFSHβ gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHβ 5'FR (~3.5 kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article.

Gonadotropins in European sea bass: Endocrine roles and biotechnological applications

“…Gnrh had no effect on fshb levels, but up-regulated pituitary lhb and cga expression (Mateos et al, 2002). Moreover, treatment with E2 inhibited the expression of the fshb gene in pituitary cells cultured in vitro (Muriach et al, 2014), showing that the negative feedback of E2 observed in vivo is acting, at least in part, directly at pituitary level. Regarding the transcriptional control of sea bass gonadotropin receptor genes, some studies performed on the promoter region of fshr have shown the existence of functional tandem binding sites for the transcription factors Sp1/Sp3 (Crespo et al, 2012) and the presence of an E-box where Usf2 can bind to activate gene expression (Lan-Chow-Wing et al, 2014).…”

Direct and Indirect Effects of Sex Steroids on Gonadotrope Cell Plasticity in the Teleost Fish Pituitary

“…SRE are short, palindromic nucleotide sequences in target genes where steroid receptors bind to regulate transcription of those genes. Full-site and half-site estrogen response elements (ERE) have been identified upstream of the lhb gene in chinook salmon ( 298 , 299 ), but only half-site EREs have been found in both fshb and lhb genes in Nile tilapia [ lhb ( 300 ); fshb ( 301 )] and goldfish [ lhb ( 80 ); fshb ( 302 )], and the fshb gene in chinook salmon ( 303 ) and sea bass ( 304 ). A half-site androgen response element (ARE) was also been identified upstream of fhb in sea bass ( 304 ).…”

“…Full-site and half-site estrogen response elements (ERE) have been identified upstream of the lhb gene in chinook salmon ( 298 , 299 ), but only half-site EREs have been found in both fshb and lhb genes in Nile tilapia [ lhb ( 300 ); fshb ( 301 )] and goldfish [ lhb ( 80 ); fshb ( 302 )], and the fshb gene in chinook salmon ( 303 ) and sea bass ( 304 ). A half-site androgen response element (ARE) was also been identified upstream of fhb in sea bass ( 304 ). Using an in vitro reporter assay, the ricefield eel lhb promoter was activated highly by E2, and to a lesser extent by T and 11-KT, indicating the presence of functionally active ERE and ARE in the lhb promoter.…”

Control of Transcriptional Repression of the Vitellogenin Receptor Gene in Largemouth Bass (Micropterus Salmoides) by Select Estrogen Receptors Isotypes

“…HEK293 (human embryonic kidney) cells were cultured in Dubelcco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal bovine serum (FBS), penicillin-streptomycin solution, and L-glutamine. These cells were employed as they are devoid of endogenous Esrs and have served as a model in vitro system for assessing promoter activity of other fish species (Muriach et al, 2014;Rebl et al, 2011;Rebl et al, 2014). HEK cells were seeded at a density of 2 x 10 5 cells/well in 24-well plates and transfected with 1000 ng of pGL3-empty vector or Vtgr-Luc constructs, and 200 ng of TKrenilla using Fugene 6.…”