Murine Relaxin-Like Factor Promoter: Functional Characterization and Regulation by Transcription Factors Steroidogenic Factor 1 and DAX-1

Abstract

The gene for mouse relaxin-like factor (RLF), a member of the insulin/IGF/relaxin family of hormones, appears to be predominantly expressed in testicular Leydig cells. Mice deficient in RLF have revealed a role for this peptide in testicular descent, but the regulatory mechanisms of its function are still insufficiently characterized. In the present study we showed that the RLF promoter was active in both mLTC-1 Leydig cells and luteinized KK-1 granulosa tumor cells. Interestingly, the activity of the RLF promoter as well as the expression of endogenous RLF correlated with the amount of steroidogenic factor 1 (SF-1) expression in the four cell lines tested. The highest transcriptional activity (29-fold over promoterless plasmid) was detected in mLTC-1 using the 188-bp promoter fragment immediately 5' of the CAP site, containing three consensus sequences for SF-1 binding. However, the promoter fragments including the 188-bp promoter also showed significant SF-1-independent promoter activity in both mLTC-1 and KK-1 cells, 8-fold induced over the promoterless construct. Mutagenesis studies showed that all three SF-1-binding sites were needed to obtain maximal SF-1-dependent trans-activation. The most distal SF-1-binding site at position -144 to -136 showed the highest affinity toward SF-1, but the promoter fragments, including the SF-1-binding site at position -115 to -107, showed the strongest response to SF-1 in terms of transcriptional activation. Moreover, DAX-1 inhibited RLF promoter activity in mLTC-1 Leydig tumor cells and totally abolished SF-1-dependent RLF expression in nonsteroidogenic HEK-293 cells. DAX-1 especially inhibited binding of SF-1 to the binding motifs locating at positions -64 to -56 and -115 to -107, whereas no decrease was seen in the expression of SF-1. Taken together, these observations suggest that the 188-bp RLF promoter includes elements for both SF-1-dependent and -independent gene expression in steroidogenic cells. The data, furthermore, indicate differential binding affinities for the three SF-1 binding motifs toward SF-1, of which the motif locating at position -115 to -107 was the most critical for the promoter activity.

Orphan Nuclear Receptor Nur77 Induces Zinc Finger Protein GIOT-1 Gene Expression, and GIOT-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor SF-1 via Recruitment of HDAC2

“…SF-1 is expressed throughout the zones of the adrenal cortex, testis, ovary, hypothalamus, and anterior pituitary (15), where it regulates a number of genes involved in the biosynthesis of steroid hormones (16 -19) and modulates the genes encoding the insulin/insulin-like growth factor/relaxin family of hormones (20). Recently, it has been reported that NR5A members, SF-1 and human LRH-1, bind phosphatidylinositol second messengers and that an endogenous ligand is required for their maximal activity (21)(22)(23).…”

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells

“…Previous cell culture experiments have shown that the sequence from -690 to +4 of Insl3 gene was able to direct expression of reporter constructs in Leydig cell lines (Zimmermann et al, 1998;Koskimies et al, 2002). In order to determine whether this part of the promoter is also sufficient in vivo to direct tissue-and cell-specific expression, transgenic mice were generated.…”

“…Analyses of the mouse Insl3 promoter in vitro predicted that the proximal region of Insl3 promoter might be sufficient for targeted expression of reporter gene in Leydig cells (Zimmermann et al, 1998;Koskimies et al, 2002). To determine the regulatory sequences of Insl3, which are required for Leydig cell-specific expression of reporter gene in vivo and for stability of transgenic RNA in Leydig cells, two transgenic constructs were designed using the 5´ flanking region of Insl3 (-690 to +4) fused to the human insulin gene (hIns).…”

The INSL3 gene is a direct target for the orphan nuclear receptor, COUP-TFII, in Leydig cells

“…6) indicate that COUP-TFII may act in association with other DNA-bound transcription factors and as the DR0-like element (K103/K91 bp) is in proximity to binding sites for the nuclear receptor SF1 (Zimmermann et al 1998, Koskimies et al 2002, one of which is located within the DR3 element (K151/K135 bp), we tested the possibility that these two nuclear receptors might cooperate to modulate Insl3 promoter activity. Co-transfections in MA-10 Leydig cells revealed that COUP-TFII and SF1 individually activate the K1087 bp Insl3 promoter about fivefold while both nuclear receptors in combination had additive effects (left panel in Fig.…”

“…To date, only two transcription factors have been shown to regulate Insl3 promoter activity in Leydig cells: the nuclear receptors steroidogenic factor 1 (SF1, Ad4BP, NR5A1) and NUR77 (NGFI-B, NR4A1) (Zimmermann et al 1998, Koskimies et al 2002, Sadeghian et al 2005, Tremblay & Robert 2005, Robert et al 2006. As these nuclear receptors are co-expressed in other cell types that do not produce INSL3, other transcription factors must participate in Insl3 transcription in Leydig cells.…”