BONE MORPHOGENETIC PROTEIN 2 STIMULATES INHIBITOR OF DNA BINDING 3 TRANSCRIPTION IN GONADOTROPE CELLS

Follicle-stimulating hormone beta subunit (FSHb) expression is regulated by proteins in the transforming growth factor beta (TGFb) superfamily, in particular the activins and inhibins. Recently, we reported that other TGFb ligands, the bone morphogenetic proteins (BMPs) stimulate FSHb transcription alone and in synergy with activins. The mechanisms mediating BMP2's effects are not yet known. Therefore, we used Affymetrix GeneChip analysis to identify BMP2 target genes in gonadotropes. Treatment of LbT2 cells with 50 ng/ml BMP2 for 24 h stimulated >2-fold increases in mRNA levels of 25 genes on 430 v.2.0 arrays while down-regulating the levels of six transcripts by greater than two-fold. Of the up-regulated genes, we took particular interest the inhibitors of DNA binding (Id). Id proteins are negative regulators of basic helix-loop-helix transcription factors and have been implicated as effectors of BMP signalling in a variety of cellular systems. BMP2 stimulated increases in Id1, 2, and 3 on the arrays, and this was confirmed by northern blot analyses. Increases in Id1 mRNA levels reflect increases in gene transcription and the underlying signalling mechanisms have been elucidated. The mechanisms through which BMPs regulate Id2 and Id3 expression, however, have not been reported. Here, we examined transcriptional regulation of the murine Id3 gene. We cloned approximately 0.9 kb of the murine Id3 5′ flanking region for use in reporter assays. This promoter fragment was both time- and dose-dependently stimulated by BMP2. BMP2 signals preferentially through complexes of the type II receptor, Bmpr2, and one of three type I receptors, ALK2, 3, or 6. All of these receptors are expressed in LbT2 cells. To determine the relevant signalling receptor in this system, we used siRNAs to knockdown expression of each of the type I receptors. The ALK3 siRNA dosedependently attenuated BMP2 stimulated Id3 promoter activity, whereas the ALK2 and ALK6 siRNAs were without effect. These data suggest that BMP2 signals through the type I receptor, ALK3, to stimulate Id3 expression. ALK3 can stimulate the phosphorylation of the intracellular signalling proteins, Smads 1, 5, and 8, which act in concert with Smad4 to regulate target gene transcription. We previously showed that all of these Smad proteins are expressed and phosphorylated by BMP2 in LbT2 cells. Here, we observed that over-expression of Smads 1 and 5, in combination with Smad4, significantly potentiated the effects of BMP2 on Id3 promoter activity. The effects with Smad8 were significantly less pronounced. Thus, BMP2 may signal through complexes of Smad1/4 or Smad5/4 to regulate transcription. To determine how and where BMP2 regulates the Id3 promoter, we used 5′ deletions of the promoter and mapped the BMP2 responsive region. Gel shift analyses identified the presence of a specific DNA binding protein complex within this promoter region whose intensity might be increased by BMP2 treatment. We are currently identifying the specific cis-element to which this complex binds and determining its role in the BMP responsiveness of the promoter. In summary, our data show that BMP2 may directly regulate Id3 transcription through a Smad1/5/4- dependent pathway downstream of the type I receptor, ALK3, in gonadotrope cells. Up-regulation of Id3 expression may mediate effects of BMP2 on FSHb transcription and we are currently testing this hypothesis. This work was supported by NIH grant HD47794 to DJB. (poster)

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