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

Synthesis of FSH by the anterior pituitary is regulated by activin, a member of the TGFβ 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 FSHβ promoter in a pituitary-derived cell line LβT2. Here, we used RNA interference technology to independently decrease the expression of Smad proteins in LβT2 cells to further investigate Smad2 and Smad3 roles in activin-dependent regulation of the FSHβ promoter. Down-regulation of Smad2 protein by small interfering RNA duplexes affects only basal transcription of FSHβ, whereas decreased expression of Smad3 abrogates activin-mediated stimulation of FSHβ 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 FSHβ 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 FSHβ 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 FSHβ promoter. Taken together, these results indicate that the functional differences between Smad2 and Smad3 in their ability to transactivate the rat FSHβ promoter lie primarily within the MH1 domain and involve structural motifs that affect DNA binding

MIP/Aquaporin 0 Represents a Direct Transcriptional Target of PITX3 in the Developing Lens

The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations

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

Synthesis of FSH by the anterior pituitary is regulated by activin, a member of the TGFβ 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 FSHβ promoter in a pituitary-derived cell line LβT2. Here, we used RNA interference technology to independently decrease the expression of Smad proteins in LβT2 cells to further investigate Smad2 and Smad3 roles in activin-dependent regulation of the FSHβ promoter. Down-regulation of Smad2 protein by small interfering RNA duplexes affects only basal transcription of FSHβ, whereas decreased expression of Smad3 abrogates activin-mediated stimulation of FSHβ 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 FSHβ 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 FSHβ 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 FSHβ promoter. Taken together, these results indicate that the functional differences between Smad2 and Smad3 in their ability to transactivate the rat FSHβ promoter lie primarily within the MH1 domain and involve structural motifs that affect DNA binding