Smad3 Mediates Activin-Induced Transcription of Follicle-Stimulating Hormone β-Subunit Gene

Abstract

Synthesis of FSH by the anterior pituitary is regulated by activin, a member of the FSH(beta) superfamily of ligands. Activin signals through a pathway that involves the activation of the transcriptional coregulators Smad2 and Smad3. Previous work from our laboratory demonstrated that Smad3, and not Smad2, is sufficient for stimulation of the rat FSH(beta) promoter in a pituitary-derived cell line L(beta)T2. Here, we used RNA interference technology to independently decrease the expression of Smad proteins in L(beta)T2 cells to further investigate Smad2 and Smad3 roles in activin-dependent regulation of the FSHbeta promoter. Down-regulation of Smad2 protein by small interfering RNA duplexes affects only basal transcription of FSH(beta), whereas decreased expression of Smad3 abrogates activin-mediated stimulation of FSH(beta) transcription. Although highly related, Smad2 and Smad3 differ in their Mad homolog (MH) 1 domains, where the Smad2 protein contains two additional stretches of amino acids that prevent this factor from binding to DNA. We investigated whether these structural features contribute to differential FSH(beta) transactivation by Smad2 and Smad3. A variety of Smad chimera constructs were generated and used in transient transfection studies to address this question. Only cotransfection of chimera constructs that contain the MH1 domain of Smad3 results in activin-mediated stimulation of the rat FSH(beta) promoter. Furthermore, the insertion of Smad2 loops into Smad3 protein renders it inactive, suggesting that DNA binding is necessary for Smad3-mediated stimulation of the rat FSH(beta) promoter. Taken together, these results indicate that the functional differences between Smad2 and Smad3 in their ability to transactivate the rat FSH(beta) promoter lie primarily within the MH1 domain and involve structural motifs that affect DNA binding.

Acute regulation of murine follicle-stimulating hormone β subunit transcription by activin A

“…Activin A stimulates the rapid phosphorylation and nuclear translocation of the R-Smads, Smad2 and Smad3 (Bernard 2004, Suszko et al 2005. These proteins partner with the co-SMAD, SMAD4, to form complexes that can bind to a palindromic SBE in the proximal promoter at 266/ 259.…”

“…For example, depletion of Smad2 or Smad3 protein levels by RNAi attenuates but does not completely block effects of 24 h activin A on Fshb transcription (Fig. 2) (Bernard 2004, Suszko et al 2005. Also, mutation of the SBE in the mouse promoter abrogates the effects of over-expressed SMADs and 4 h activin A, but 24 h activin A can still significantly stimulate transcription (Fig.…”

“…We employed this approach here to probe the involvement of Smads 2, 3 and 4 in mouse Fshb transcription. To overcome previous difficulties in over-expressing SMAD2 in L T2 cells (Bernard 2004, Gregory et al 2005, while avoiding a need to change our culturing conditions (Suszko et al 2005), we used Flag-tagged full-length human SMAD2 in pCAGGS (Dunn et al 2004), which contains the chicken -actin promoter (Niwa et al 1991). Using this construct we were able to over-express full-length SMAD2 in L T2 cells at levels comparable to over-expressed SMAD3 from the weaker CMV promoter (Fig.…”

Transforming growth factor-β1 and activin A generate antiproliferative signaling in thyroid cancer cells

“…Our study showed that TPC expresses SMADs mRNA and that RNAi targeting TGF-b1 mRNA and ActA mRNA inhibited gene expression of stimulatory SMADs, SMAD2, SMAD3, and SMAD4, thereby suggesting that both TGF-b1 and ActA modulate the expression of their mediators and generate signaling that acts cooperatively in thyroid cancer. Although SMAD2 and SMAD3 share a high percentage of homology and both are stimulated by the complex TGF-b/receptor or activin/ receptor, many in vitro studies have shown the differential participation of SMAD2 and SMAD3 in transducing the signal generated by TGF-b/activin in specific cell types (Kretschmer et al 2003, Suszko et al 2005. In SMAD knockout mice, this differential role of each stimulatory SMAD is evident by observation that gene inactivation of either SMAD2 or SMAD3 produces distinct phenotypes (Chang et al 2002).…”

The structures that underlie normal reproductive function

“…The solution structure of the Pitx2 homeodomain is the first to characterize a native K50 class homeodomain and elaborates on the specificity of this protein with its consensus DNA binding sequence (Chaney et al, 2005). Smad3/4, along with Pitx2, activate transcription of FSHβ (Suszko et al, 2005), which acts in an endocrine fashion to stimulate follicle development (Fig 2-4). While the full cooperative effects of the SMAD3/4 complex and Pitx2 are being unraveled, these advances demonstrate the structural intricacies that are maintained in the activin signaling pathway.…”