Bone morphogenetic proteins (BMPs) are potent inhibitors of myoblast differentiation and inducers of bone formation both in vivo and in vitro. Expression of Id1, a negative regulator of basic helix-loop-helix transcription factors, is up-regulated by BMPs and contributes to the antimyogenic effects of this family of cytokines. In this report, we have identified a specific BMP-2 immediate early response enhancer in the human Id1 gene. Transcriptional activation of the enhancer was increased by overexpression of BMP-responsive Smads, and Smad4 and was completely abrogated in Smad4-deficient cells. Deletion analysis demonstrates that the responsive region is composed of two separate DNA binding elements, a set of overlapping GC boxes, which bind BMP-regulated Smads upon BMP stimulation, and three repeats of CAGAC boxes. Gel shift and oligonucleotide pull-down assays demonstrated that these two types of motifs were capable of binding their corresponding Smads. However, deletion or mutation of either DNA binding element was nonadditive, since disruption of either GC or CAGAC boxes resulted in complete or severe loss of BMP-2 responsiveness. These data suggest the simultaneous requirement of two independent DNA binding elements to allow functional cooperativity of BMP-regulated Smads and Smad4 in BMP-activated gene promoters.

Bone morphogenetic proteins (BMPs) constitute a family of multifunctional growth and differentiation factors structurally related to transforming growth factor-␤. BMPs were first identified by their osteoinductive effects, inducing ectopic bone formation when implanted in skeletal muscle, and have an important role as regulators of skeletal development in vivo. In vitro, BMP-2 is able to transdifferentiate myogenic C2C12 cells into the osteoblastic phenotype. In this report, we show that the osteoinductive effects of BMP-2 in C2C12 cells are mediated by bone morphogenetic protein receptor type-IA in combination with both activin receptor type II and bone morphogenetic protein receptor type II. We also analyzed the expression levels of nuclear protooncogenes to understand early transcriptional events induced by BMP-2. We show that junB is an immediate early gene induced by BMP-2 and transforming growth factor-␤. BMP-2 induces transcriptional activation of JunB expression as early as 30 min after ligand addition, reaching maximal levels after 90 min. Increase of JunB mRNA correlates with a higher AP-1 binding activity. Furthermore, ectopic overexpression of JunB is sufficient to inhibit expression of myoblast differentiation markers in C2C12 cells. These data, taken together, show the involvement of JunB in the early steps of inhibition of myogenic differentiation induced by transforming growth factor-␤ family members.

Transforming growth factor-L L (TGF-L L) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc-finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF-L L in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF-L L effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl-2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co-expression of Bcl-2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro-apoptotic properties of TGF-L L.z 1999 Federation of European Biochemical Societies.