Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype

Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na⁺/H⁺ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology.     

Profilin1 Regulates Sternum Development and Endochondral Bone Formation

Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration     

The Factor VII-activating Protease (FSAP) Enhances the Activity of Bone Morphogenetic Protein-2 (BMP-2)

Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg²⁸²↓Gln²⁸³), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg²⁸⁹↓Lys²⁹⁰). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg²⁸⁹↓Lys²⁹⁰). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases.     

MicroRNA-22 Is a Master Regulator of Bone Morphogenetic Protein-7/6 Homeostasis in the Kidney

Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.     

Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling,Promote Chondrogenesis,and Reveal a BMP2-like Structure

Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.     

Bone Morphogenetic Protein functions as a context-dependent angiogenic cue in vertebrates

Bone Morphogenetic Protein (BMP) signaling has been implicated in diverse biological processes. Although how BMP signaling regulates behaviors of endothelial cells during angiogenesis are not fully understood, increasing evidence indicate functions of BMP signaling components are essential in developmental and pathological angiogenesis. Here we review recent advances in delineating the functions of BMP signaling during angiogenesis. In addition, we discuss downstream pathways that transduce BMP signaling in endothelial cells, and factors that modulate BMP signaling response in endothelial cells. Finally, we provide recent insight on how BMP signaling functions as a context dependent angiogenic cue.     

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C₂-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C₂-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.     

Hedgehog-Gli Activators Direct Osteo-chondrogenic Function of Bone Morphogenetic Protein toward Osteogenesis in the Perichondrium

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1^−/− fetuses, and the phenotype was more severe in Gli1^−/−;Gli2^−/− newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.     

Bone morphogenetic proteins: a critical review

Bone Morphogenetic Proteins (BMPs) are potent growth factors belonging to the Transforming Growth Factor Beta superfamily. To date over 20 members have been identified in humans with varying functions during processes such as embryogenesis, skeletal formation, hematopoesis and neurogenesis. Though their functions have been identified, less is known regarding levels of regulation at the extracellular matrix, membrane surface, and receptor activation. Further, current models of activation lack the integration of these regulatory mechanisms. This review focuses on the different levels of regulation, ranging from the release of BMPs into the extracellular components to receptor activation for different BMPs. It also highlights areas in research that is lacking or contradictory.     

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.     

Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis

BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv^−/−) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv^−/− mice do not lack BMP6. Furthermore, iron depletion in Hjv^−/− mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv^−/− mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling.     

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.     

Identification of a Lysosomal Pathway Regulating Degradation of the Bone Morphogenetic Protein Receptor Type II

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease.