BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.     

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.     

A TGFβ-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

The tongue is a muscular organ and plays a crucial role in speech, deglutition and taste. Despite the important physiological functions of the tongue, little is known about the regulatory mechanisms of tongue muscle development. TGFβ family members play important roles in regulating myogenesis, but the functional significance of Smad-dependent TGFβ signaling in regulating tongue skeletal muscle development remains unclear. In this study, we have investigated Smad4-mediated TGFβ signaling in the development of occipital somite-derived myogenic progenitors during tongue morphogenesis through tissue-specific inactivation of Smad4 (using Myf5-Cre;Smad4(flox/flox) mice). During the initiation of tongue development, cranial neural crest (CNC) cells occupy the tongue buds before myogenic progenitors migrate into the tongue primordium, suggesting that CNC cells play an instructive role in guiding tongue muscle development. Moreover, ablation of Smad4 results in defects in myogenic terminal differentiation and myoblast fusion. Despite compromised muscle differentiation, tendon formation appears unaffected in the tongue of Myf5-Cre;Smad4(flox/flox) mice, suggesting that the differentiation and maintenance of CNC-derived tendon cells are independent of Smad4-mediated signaling in myogenic cells in the tongue. Furthermore, loss of Smad4 results in a significant reduction in expression of several members of the FGF family, including Fgf6 and Fgfr4. Exogenous Fgf6 partially rescues the tongue myoblast fusion defect of Myf5-Cre;Smad4(flox/flox) mice. Taken together, our study demonstrates that a TGFβ-Smad4-Fgf6 signaling cascade plays a crucial role in myogenic cell fate determination and lineage progression during tongue myogenesis.     

Osteogenic properties of human myogenic progenitor cells

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.     

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-β superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-β pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

Heparin mimicking polymer promotes myogenic differentiation of muscle progenitor cells

Heparin and heparan sulfate mediated basic fibroblast growth factor (bFGF) signaling plays an important role in skeletal muscle homeostasis by maintaining a balance between proliferation and differentiation of muscle progenitor cells. In this study we investigate the role of a synthetic mimic of heparin, poly(sodium-4-styrenesulfonate) (PSS), on myogenic differentiation of C2C12 cells. Exogenous supplementation of PSS increased the differentiation of C2C12 cells in a dose-dependent manner, while the formation of multinucleated myotubes exhibited a nonmonotonic dependence with the concentration of PSS. Our results further suggest that one possible mechanism by which PSS promotes myogenic differentiation is by downregulating the mitogen activated extracellular regulated signaling kinase (MAPK/ERK) pathway. The binding ability of PSS to bFGF was found to be comparable to heparin through molecular docking calculations and by native PAGE. Such synthetic heparin mimics could offer a cost-effective alternative to heparin and also reduce the risk associated with batch-to-batch variation and contamination of heparin.     

Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death

Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.     

BMP4 regulates vascular progenitor development in human embryonic stem cells through a smad‐dependent pathway

The signals that direct pluripotent stem cell differentiation into lineage‐specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum‐free condition, hESCs sequentially differentiated into CD34+CD31?, CD34+CD31+, and then CD34?CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFβ suppressed BMP4‐induced CD34+CD31+ cell development, and promoted CD34+CD31? cells that failed to give rise to either endothelial or smooth muscle cells. The BMP‐Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad‐dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells. J. Cell. Biochem. 109: 363–374, 2010. © 2009 Wiley‐Liss, Inc.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

Interplay between BMP4 and IL-7 in human intrathymic precursor cells

Bone morphogenetic proteins (BMPs) play a pivotal role during vertebrate embryogenesis and organogenesis, and have also been described to function in regulating cell fate and determination in self-renewing tissues in adults. Recent results have demonstrated that the different components of the BMP2/4 signaling pathway are expressed in the human thymus. In this study, we provide evidence that BMP4 and IL-7 interplay is important in the maintenance of the human thymic progenitor population. Intrathymic CD34⁺ cells express BMP receptors (BMPRIA, BMPRIB, ActRIA, BMPRII), signal transduction molecules (Smad1, 5, 8 and 4), and produce BMP4. Neutralization of endogenous BMP4 by treatment with the antagonist Noggin reduces thymic precursor cell survival, and the addition of exogenous BMP4 decreases their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with BMP4 inhibits cell expansion, arrests thymocyte differentiation, and leads to the accumulation of human CD34⁺ precursor cells. This effect is mainly attributed to the ability of BMP4 to counteract the IL-7-induced proliferation and differentiation of CD34⁺ cells. BMP4 down-regulates in the precursor cell population the expression of CD127 and inhibits the IL-7-dependent STAT5 phosphorylation. In addition, BMP signaling is promoted by IL-7. Our results also demonstrate that in thymic progenitors BMPs act downstream of Sonic Hedgehog, previously described to function as a maintenance factor for human intrathymic CD34⁺ precursor cells.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

Sox9 Represses α-Sarcoglycan Gene Expression in Early Myogenic Differentiation

Alpha sarcoglycan (α-SG) is highly expressed in differentiated striated muscle, and its disruption causes limb-girdle muscular dystrophy. Accordingly, the myogenic master regulator MyoD finely modulates its expression. However, the mechanisms preventing α-SG gene expression at early stages of myogenic differentiation remain unknown. In this study, we uncovered Sox9, which was not previously known to directly bind muscle gene promoters, as a negative regulator of α-SG gene expression. Reporter gene and chromatin immunoprecipitation assays revealed three functional Sox-binding sites that mediate α-SG promoter activity repression during early myogenic differentiation. In addition, we show that Sox9-mediated inhibition of α-SG gene expression is independent of MyoD. Moreover, we provide evidence suggesting that Smad3 enhances the repressive activity of Sox9 over α-SG gene expression in a transforming growth factor-β-dependent manner. On the basis of these results, we propose that Sox9 and Smad3 are responsible for preventing precocious activation of α-SG gene expression during myogenic differentiation.     

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.     

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.     

Genes that control the development of migrating muscle precursor cells

Skeletal muscles in vertebrates, despite their functional and biochemical similarities, are generated via diverse developmental mechanisms. A major subclass of hypaxial muscle groups is derived from long-range migrating progenitor cells that delaminate from the dermomyotome. The development of this lineage is controlled by Pax3, the c-Met tyrosine kinase receptor, its ligand SF/HGF (scatter factor/hepatocyte growth factor) and the homeobox factor Lbx1. These molecules are essential for establishment of the precursor pool, delamination, migration and target finding. Progress has been made in understanding patterning of the muscles, which requires a precise control of proliferation and differentiation of myogenic precursor cells.