Smad3 mediates activin-induced transcription of follicle-stimulating hormone beta-subunit gene.
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.
Suszko
Magdalena I
MI
Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208 USA.
Balkin
Daniel M
DM
Chen
Yan
Y
Woodruff
Teresa K
TK
D021581:Active Transport, Cell Nucleus; D028341:Activins / Q000494:pharmacology*; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D037201:Follicle Stimulating Hormone, beta Subunit; D011993:Recombinant Fusion Proteins; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D015534:Trans-Activators; D028341:Activins