Smad1, Smad5 and Smad9 (also known as Smad8) are activated by phosphorylation by bone morphogenetic protein (BMP)-bound type I receptor kinases. We examined the role of Smad1, Smad5, and Smad9 by creating constitutively active forms (SmadDVD). Transcriptional activity of Smad9DVD was lower than that of Smad1DVD or Smad5DVD, even though all three SmadDVDs associated with Smad4 and bound to the target DNA. The linker region of Smad9 was sufficient to reduce transcriptional activity. Smad9 expression was increased by the activation of BMP signaling, similar to that of inhibitory Smads (I-Smads), and Smad9 reduced BMP activity. In contrast to I-Smads, however, Smad9 did not inhibit the type I receptor kinase and suppressed the constitutively active Smad1DVD. Smad9 formed complexes with Smad1 and bound to DNA but suppressed the transcription of the target gene. Taken together, our findings suggest that Smad9 is a new type of transcriptional regulator in BMP signaling.

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

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Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).

Bone morphogenetic proteins (BMPs) inhibit myogenesis and induce osteoblastic differentiation in myoblasts. They also induce the transcription of several common genes, such as Id1, Id2 and Id3, in various cell types. We have reported that a GC-rich element in the Id1 gene functions as a BMP-responsive element (BRE) that is regulated by Smads. In this study, we analyzed and identified BREs in the 5'-flanking regions of the mouse Id2 and Id3 genes. The core GGCGCC sequence was conserved among the BREs in the Id1, Id2 and Id3 genes and was essential for the response to BMP signaling via Smads. We found a novel BRE on mouse chromosome 13 at position 47,723,740-47,723,768 by searching for conserved sequences containing the Id1 BRE. This potential BRE was found in the 5'-flanking region of a novel gene that produces a non-coding transcript, termed BMP-inducible transcript-1 (BIT-1), and this element regulated the expression of this gene in response to BMP signaling. We found that BIT-1 is expressed in BMP target tissues such as the testis, brain, kidney and cartilage. These findings suggest that the transcriptional induction of the Ids, BIT-1 and additional novel genes containing the conserved BRE sequence may play an important role in the regulation of the differentiation and/or function of target cells in response to BMPs.

Smads 1/5/8 transduce the major intracellular signaling of bone morphogenetic proteins (BMPs). In the present study, we analyzed Smad1-binding proteins in HEK293T cells using a proteomic technique and identified the protein, zinc-finger, RAN-binding domain-containing protein 2 (ZRANB2). Zranb2 interacted strongly with Smad1, Smad5, and Smad8 and weakly with Smad4. The overexpression of Zranb2 inhibited BMP activities in C2C12 myoblasts in vitro, and the injection of Zranb2 mRNA into zebrafish embryos induced weak dorsalization. Deletion analyses of Zranb2 indicated that the serine/arginine-rich (SR) domain and the glutamine-rich domain were required for the inhibition of BMP activity and the interaction with Smad1, respectively. Zranb2 was found to be localized in the nucleus; however, the SR domain-deleted mutant localized to the cytoplasm. The knockdown of endogenous Zranb2 in C2C12 cells enhanced BMP activity. Zranb2 suppressed Smad transcriptional activity without affecting Smad phosphorylation, nuclear localization, or DNA binding. Taken together, these findings suggested that Zranb2 is a novel BMP suppressor that forms a complex with Smads in the nucleus.

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