Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation.     

Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.     

Bone morphogenetic protein signaling in articular chondrocyte differentiation

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.     

Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.     

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.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

Divergence and convergence of TGF-beta/BMP signaling

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.