The RING domain of PIASy is involved in the suppression of bone morphogenetic protein-signaling pathway

Bone morphogenetic proteins (BMPs) play central roles in differentiation, development, and physiologic tissue remodeling. Recently, we have demonstrated that a protein inhibitor of activated STAT, PIASy, suppresses TGF-beta signaling by interacting with Sma and MAD-related protein 3 (Smad3). In this study, we examined a PIASy-dependent inhibitory effect on BMP signaling. PIASy expression was induced by BMP-2 stimulation and suppressed BMP-2-dependent Smad activity in hepatoma cells. Furthermore, BMP-2-regulated Smads directly bound to PIASy. We also demonstrated that the RING domain of PIASy played an important role in PIASy-mediated suppression of Smad activity. We here provide evidence that the inhibitory action of PIASy on BMP-regulated Smad activity was due to direct physical interactions between Smads and PIASy through its RING domain.     

Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.     

Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-beta type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

XMAN1, an inner nuclear membrane protein,antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.     

Cell fate specification and competence by Coco,a maternal BMP,TGFbeta and Wnt inhibitor

Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFbeta/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFbeta signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.     

Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation

TGF-beta inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-beta in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-beta signaling in adipocyte differentiation, we characterized the expression of the TGF-beta receptors, and the Smads which transmit or inhibit TGF-beta signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-beta receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-beta responses, decreased severely. Dominant negative interference with TGF-beta receptor signaling, by stably expressing a truncated type II TGF-beta receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-beta-induced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-beta was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-beta signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-beta responses, enhance the effects of TGF-beta on these cells.