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.     

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.     

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid β peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1–205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.     

Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts

The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125(FAK). We report here that activation of p125(FAK) occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2alpha/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.     

Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

Objectives

Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.

Materials and methods

Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.

Results

BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.

Conclusions

These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.     

Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3β-dependent and β-catenin-independent mechanism

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of β-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3β activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3β-dependent but β-catenin-independent mechanism.     

Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.     

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.     

The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways.

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.     

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.     

A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.     

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.     

BMP-5 expression increases during chondrocyte differentiation in vivo and in vitro and promotes proliferation and cartilage matrix synthesis in primary chondrocyte cultures

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short-ear (se) mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 loss-of-function mutations is discussed.     

Activation of the ERK pathway in osteoblastic cells, role of gremlin and BMP-2

Gremlin is a glycoprotein that binds and antagonizes the actions of bone morphogenetic proteins (BMPs) -2, -4, and -7. Gremlin appears to activate the extracellular regulated kinase (ERK) pathway in endothelial and tumor cells, and as a consequence to have direct cellular effects. To determine whether gremlin has direct effects in osteoblasts, independent of its BMP binding activity, we examined its effects in ST-2 murine stromal cell lines and in primary cultures of murine calvarial osteoblasts. Gremlin did not activate Signaling mothers against decapentaplegic (Smad), and suppressed the BMP-2 induced Smad 1/5/8 phosphorylation and the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3, confirming its BMPs antagonizing activity. Neither gremlin nor BMP-2 induced ERK 1/2 activation in ST-2 cells or calvarial osteoblasts. Moreover, slight changes in culture conditions induced the phosphorylation of ERK independent from BMP or gremlin exposure. In conclusion, gremlin inhibits BMP-2 signaling and activity, and does not have independent actions on ERK signaling in osteoblasts. Consequently, gremlin activity in osteoblasts can be attributed only to its BMP antagonizing effects.