BMP Signaling Regulates Bone Morphogenesis in Zebrafish through Promoting Osteoblast Function as Assessed by Their Nitric Oxide Production.

Bone morphogenetic proteins (BMPs) control many developmental and physiological processes, including skeleton formation and homeostasis. Previous studies in zebrafish revealed the crucial importance of proper BMP signaling before 48 h post-fertilization (hpf) for cartilage formation in the skull. Here, we focus on the involvement of the BMP pathway between 48 and 96 hpf in bone formation after 96 hpf. Using BMP inhibitors and the expression of a dominant-negative BMP receptor, we analyze whether the loss of BMP signaling affects osteoblastogenesis, osteoblast function and bone mineralization. To this end, we used the transgenic zebrafish line Tg(osterix:mCherry), detection of nitric oxide (NO) production, and alizarin red staining, respectively. We observed that inhibition of BMP signaling between 48 and 72 hpf led to a reduction of NO production and bone mineralization. Osteoblast maturation and chondrogenesis, on the other hand, seemed unchanged. Osteoblast function and bone formation were less affected when BMP signaling was inhibited between 72 and 96 hpf. These results suggest that for the onset of bone formation, proper BMP signaling between 48 and 72 hpf is crucial to ensure osteoblast function and ossification. Furthermore, detection of NO in developing zebrafish larvae appears as an early indicator of bone calcification activity.

Drosophila S6 Kinase like inhibits neuromuscular junction growth by downregulating the BMP receptor thickveins.

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells.

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-ß signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell-cell junctions and instead activates Ras homologue gene family A (RhoA)-Rho-associated protein kinase (ROCK)-canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest-like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.

Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells.

Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

Induced migration of dental pulp stem cells for in vivo pulp regeneration.

Dental pulp has intrinsic capacity for self-repair. However, it is not clear whether dental pulp cells can be recruited endogenously for regenerating pulp tissues, including mineralizing into dentin. This work is based on a hypothesis that dental pulp stem/progenitor cells can be induced to migrate by chemotactic cytokines and act as endogenous cell sources for regeneration and mineralization. Dental stem cells (DSCs) were isolated from adult human tooth pulp and seeded on the surfaces of 3D collagen gel cylinders that were incubated in chemically defined media with stromal-derived factor-1α (SDF1), basic fibroblast growth factor (bFGF), or bone morphogenetic protein-7 (BMP7). Significantly more cells were recruited into collagen gel by SDF1 or bFGF than without cytokines in 7 days, whereas BMP7 had little effect on cell recruitment. BMP7, however, was highly effective, equally to dexamethasone, in orchestrating mineralization of cultured DSCs. Cell membrane receptors for SDF1, bFGF, and BMP7 were up-regulated in treated DSCs. Upon in vivo delivery, bFGF induced re-cellularization and re-vascularization in endodontically treated human teeth implanted into the dorsum of rats. Thus, endogenous dental pulp cells, including stem/progenitor cells, may be recruited and subsequently differentiated by chemotaxis of selective cytokines in the regeneration of dental pulp.

Bone morphogenetic protein receptor expressions in the adult rat brain.

Bone morphogenetic proteins (BMP) are members of the transforming growth factor ß (TGF-ß) superfamily. BMPs exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Although BMPR expressions have been well described in the early development of the CNS, little information is available for their expressions in the adult CNS. We, thus, investigated BMPR expressions in the adult rat CNS using immunohistochemistry. Here, we show that BMPRIA, IB and II proteins are widely expressed throughout the adult CNS. Interestingly, we observed that BMPRIA, IB and II proteins are abundantly expressed in many kinds of axons. In addition, we found that BAMRIB-IR was preferentially expressed in dendrites of many neurons throughout the CNS, while BMPRIA was mainly expressed in cell bodies, showing that BMPRIA and BMPRIB are differentially targeted in a single neuron. In addition, besides abundant BMPR expressions in neurons, we exhibited BMPR expressions in astrocytes and ependymal cells. These data indicate that BMPRs are more widely expressed throughout the adult CNS than previously reported, and their continued abundant expressions in the adult brain strongly support the idea that BMPRs play pivotal roles also in the adult brain.

Canonical BMP signaling is dispensable for hematopoietic stem cell function in both adult and fetal liver hematopoiesis, but essential to preserve colon architecture.

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5, we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly, concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However, Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover, although BMP receptor expression is increased in fetal liver, fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion, canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis, despite its crucial role in the initial patterning of hematopoiesis in early embryonic development.

Bone morphogenetic protein 7 (BMP-7) increases the expression of follicle-stimulating hormone (FSH) receptor in human granulosa cells.

OBJECTIVE: To examine the effect of bone morphogenetic protein 7 (BMP-7) on FSH receptor (FSHR) expression in human granulosa cells. DESIGN: Laboratory study using human samples. SETTING: University hospital. PATIENT(S): Human granulosa cells were obtained from 60 women undergoing oocyte retrieval for IVF. INTERVENTION(S): Human granulosa cells (GCs) were cultured with recombinant BMP-7, followed by RNA extraction. MAIN OUTCOME MEASURE(S): mRNA levels of GCs were measured by real-time reverse-transcription polymerase chain reaction. RESULT(S): Bone morphogenetic protein 7 increased FSHR gene expression in human luteinized granulosa cells, whereas it decreased LH receptor gene expression. Bone morphogenetic protein 7 also increased FSH-induced cyclic adenosine monophosphate production in GCs, indicating up-regulation of the cellular response to FSH. Although BMP-7 increased gene expression of activin-betaA and -betaB in GCs, inhibition of activin function did not affect the BMP-7-induced FSHR gene expression. CONCLUSION(S): These findings provide new insight into the biologic function of BMP-7 in the human ovary and demonstrate its unique mechanism of regulating FSHR action.

Endogenous bone morphogenetic proteins in human bone marrow-derived multipotent mesenchymal stromal cells.

Primary human multipotent mesenchymal stromal cells (MSCs) are capable of self renewal or differentiation into several different lineages, including osteoblasts, chondrocytes and adipocytes. However, upon prolonged in vitro culture, MSCs tend to undergo spontaneous osteogenic differentiation. Here, we address the possible role of endogenous osteogenic bone morphogenetic proteins (BMPs) in in situ osteoblastic differentiation of human MSCs. Human MSCs consistently express biologically active BMP-2, BMP-4 and BMP-6 in addition to all BMP-activated receptors, which are functional as shown by the induction of alkaline phosphatase (ALP) activity and up-regulation of osteogenic genes (ALP, BSP1, collagen I and Runx2) following BMP-2 exposure. Since glycosaminoglycans (GAGs) have been implicated in the modulation of the osteogenic bioactivity of BMPs, we reduced sulphated cell surface GAGs by NaClO(3) treatment and found significantly reduced osteogenic gene expression and ALP activity, suggesting that this was partly due to the reduced biological activity of endogenous BMPs. Antagonising osteogenic BMP activity led to a significant reduction in the ALP activity and down-regulation of the transcription factor Runx2 associated with osteogenic development. Blocking BMP receptor type I kinase function with dorsomorphin demonstrated that endogenous osteogenesis was independent of Smad activation but was dependent on phosphatidylinositol 3-kinase (PI-3K). Inclusion of the PI-3K kinase inhibitor Ly294002 significantly reduced osteogenic gene expression and ALP activity. Spontaneous mineralisation was also abrogated following PI-3K inhibition. Thus, endogenous BMPs could contribute to spontaneous osteogenesis through Smad-independent PI-3K-dependent signalling.

Expression of bone morphogenetic proteins and receptors in porcine cumulus-oocyte complexes during in vitro maturation.

In vitro oocyte growth is the essential technology which enables oocytes to achieve maturation and acquire the competence for subsequent manipulation. There is increasing evidence that members of the transforming growth factor-beta (TGF-beta) superfamily are expressed in a variety of cell types within the ovary in a developmental stage-related manner and function as crucial factors in oocyte growth and follicular development. However, the expression of TGF-beta family members has been studied extensively in follicular compartment cells in the ovaries while poorly explored in the cumulus-oocytes complex (COC) within culture systems. Using semi-quantitative RT-PCR, we investigated the temporal and spatial expression patterns of several bone morphogenetic proteins (BMP-4, BMP-6, BMP-15 and GDF-9), as well as BMP receptors (BMPRIA, BMPRIB, BMPRII and ActRII), in porcine COCs throughout in vitro maturation (IVM). In oocytes, the transcription of BMP-6, BMP-15, GDF-9 and BMPRII were down-regulated, while BMP-4, BMPRIA and BMPRIB remained unchanged during IVM. In cumulus cells, BMP-4 mRNA expression increased significantly, while BMP-6 and ActRII was down-regulated during IVM. Nevertheless, mRNAs of BMPRIA, BMPRIB and BMPRII were constantly expressed in cumulus cells in the process. However, BMP-15 was absent in cumulus cells and ActRII was not detected in oocytes. In addition, hardly any transcription of BMP-2, BMP-5, BMP-7, ActRIA was found in porcine COCs throughout IVM. These data demonstrate a complex BMP-signaling system for member gene expression within porcine COCs during IVM and indicate the need for further functional characterization of these factors during oocyte maturation.

Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes.

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.

Inhibitory effects and target genes of bone morphogenetic protein 6 in Jurkat TAg cells.

Bone morphogenetic proteins (BMP) are multifunctional cytokines that belong to the TGF-beta superfamily. BMP have been shown to regulate haematopoietic stem cells, B lymphopoiesis and early thymocyte differentiation. In the present study we explored the role of BMP-6 in Jurkat TAg cells. BMP-6 rapidly induced phosphorylation of Smad1/5/8, p38 and ERK1/2, followed by a potent up-regulation of ID1, ID2 and ID3. ID1 and ID3 were also induced at the protein level. Genome-wide expression profiling of cells treated with BMP-6 compared to medium confirmed that ID1-ID3 were target genes of BMP-6 together with Noggin and Smad6. Furthermore, several genes involved in transcriptional regulation were also identified, including NFKBIA, HEY1, DLX2, KLF10 and early growth response 1. Stimulation with BMP-6 exerted an antiproliferative effect that was counteracted by inhibitor of DNA binding (Id)1 siRNA, indicating that Id1 is an important downstream mediator in Jurkat TAg cells. A subset of CD4(+) T cells were found to express the BMP receptors Alk-2 and Alk-3 (type I), in addition to BMPRII (type II). BMP-6 also induced phosphorylation of Smad1/5/8, followed by transcriptional increase in ID1-ID3 mRNA expression. However, we did not observe significant changes in Id protein expression in CD4(+) T cells. Altogether, the data indicate a role for BMP-6 in human T lineage cells.

BMP2, BMP4, and their receptors are expressed in the differentiating muscle tissues of mouse embryonic tongue.

To investigate the role of bone morphogenetic proteins (BMPs) in the differentiation process of skeletal muscle, we analyzed the in vivo expression of BMP2 and BMP4, of BMP receptors (BMPR) IA, IB, and II, and of activin receptors (ActR) IA, II, and IIB in mouse tongue muscle between embryonic day 11 (E11) and E17. The mRNA expression levels for BMP2 were 5-fold to 11-fold greater than those for BMP4 between E13 and E17 (P < 0.05-0.01). Expression of the BMP2, BMPRIB, ActRIA, ActRII, and ActRIIB proteins was first observed at E13. Expression of BMP2 and BMPRIB was detected in the whole area of the differentiating muscle tissues identified by immunostaining for fast myosin heavy chain (fMHC), but that of ActRIA, ActRII, and ActRIIB was detected only in the peripheral area of the differentiating muscle tissues. In the E15 tongue, all of the BMPs, BMPRs, and ActRs studied herein were expressed in the whole area of the differentiating muscle tissues identified by immunostaining for fMHC. These results suggest that BMPs play a role in the differentiation of tongue muscle tissues at E15 but have little or no effect at E13.

Expression of bone morphogenetic proteins and their receptors in the bone marrow megakaryocytes of GATA-1(low) mice: a possible role in osteosclerosis.

The mechanism of osteosclerosis associated with myelofibrosis in megakaryocyte (MK)-related myeloproliferative disorders is largely unknown. However, growth factors released from the bone marrow cells, including from MKs, have been implicated in myelofibrosis, osteosclerosis, and angiogenesis. GATA-1 is a transcription factor required for normal MK development. GATA-1 deficiency in mice (GATA-1(low)) leads to increased megakaryocytic proliferation, followed by osteosclerosis and myelofibrosis. In this study we investigated the expression of bone morphogenetic proteins (BMPs) and BMP receptors and their possible role in the development of osteosclerosis in the MKs of 12-month-old GATA-1(low) mice by immunohistochemistry, cytomorphometry, and quantitative real-time PCR. Marrow MKs from both wild-type and GATA-1(low) mice showed moderate to intense staining for BMP-2, -4, and -6 and BMPR-IA and BMPR-II, whereas splenic MKs showed no BMP immunostaining. Presence of BMP protein in the bone marrow of GATA-1(low) mice was more than that seen in controls, owing to an increased number of MKs and osteoblasts. The osteosclerosis seen in GATA-1(low) mice appeared not to be due to a reduced number of functional osteoclasts because the number of tartrate-resistant acid phosphatase-positive osteoclasts was greater in GATA-1(low) mice than in controls. Our findings demonstrate the presence of significant amounts of BMP-2, -4, and -6 along with their receptors in bone marrow MKs of WT and GATA-1(low) mice. The increased levels of BMPs appear to be a result of increased numbers of MKs in GATA-1(low) mice and may, in part, account for the stimulation of osteoblastic activity and resulting osteosclerosis.

Expression of bone morphogenic proteins and receptors at the injured growth plate cartilage in young rats.

The injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Bone morphogenic proteins (BMPs) are important for bone fracture repair, and as a step to characterize potential involvement of BMPs in bony repair of injured growth plate, expression of BMPs and receptors (BMP-R) was examined by quantitative RT-PCR and immunohistochemistry in rat injured tibial growth plate. During the inflammatory response on day 1, slightly increased expression of BMP-3, BMP-4, BMP-R1a, and BMP-R2 was observed, with immunostaining seen among inflammatory cells at the injury site. During mesenchymal infiltration and osteogenic responses on days 3-14, moderately increased expression of BMP-2, -3, -4, -7, and BMP-R1a was found, with immunostaining observed among infiltrated mesenchymal cells and differentiated osteoblasts lining bony trabeculae. During maturation phase on days 14-25, only BMP-7 was seen upregulated slightly and was localized in osteoblasts and marrow cells at the injury site. The temporospatial expression of BMPs and receptors at the injured growth plate suggests potential involvement of BMP-3 and -4 in regulating the inflammatory response or as its mediators in modulating downstream events, and BMP-2, -3, -4, and -7 in the fibrogenic and osteogenic responses, and BMP-7 in bone remodeling at the injured growth plate.

Coordinate expression of BMP-2, BMP receptors and Noggin in normal mouse spine.

The purpose of this study was to determine the localization of bone morphogenetic protein-2 (BMP-2), BMP receptors (BMPRs) and Noggin in mouse spinal tissues. The coordinate expression of these positive and negative regulators of BMP signaling may elucidate regulatory mechanisms for bone induction in the spine. Whole spines from 3-week-old mice were used and the spatial expression profiles of BMP-2, BMPR-1a, -1b, -2 and Noggin were examined using in situ hybridization. BMP-2, BMPR-1b and -2 were observed in bone marrow cells in the vertebrae, chondrocytes, hyaline cartilage cells and fibrous cells in the intervertebral discs and neurons of the spinal cord in the entire spine. BMPR-1a was also observed in these cells, but only in the cervical spine. Noggin was expressed in bone marrow cells in the vertebrae, chondrocytes and hyaline cartilage cells and fibrous cells in the intervertebral discs in the entire spine and in neurons in the spinal cord in the cervical and thoracic regions. Noggin was also expressed in the anterior longitudinal, posterior longitudinal and yellow ligaments in the cervical spine, and in the fibrous cells in the anterior longitudinal and yellow ligaments of the lumbar spine.

Expression profiles of BMP-related molecules induced by BMP-2 or -4 in muscle-derived primary culture cells.

The formation of ectopic bone in muscle following the implantation of decalcified bone matrix led to the search and eventual discovery of bone morphogenetic proteins (BMPs) in bone matrix. The precise sequence of molecular events that underpin the cellular transformation of undifferentiated mesenchymal cells into bone has not been established, and is the subject of this study. Northern and Western blot analyses were used to examine changes in gene expression of cells treated with BMP-2 or -4. The molecules, which included BMP receptors (BMPRs), Noggin (a BMP-specific antagonist), osteocalcin (OC), Smad-4, and MyoD, were examined at messenger RNA (mRNA) and protein levels. The changes in expression of these molecules were followed in mouse muscle-derived primary culture cells, and osteoblastic or nonosteoblastic embryonic cell lines. We show the early up-regulation of BMPR-1A, -2, Noggin, OC, and Smad-4 in muscle-derived primary culture cells in a dose-dependent manner in response to BMP-2 or -4. MyoD expression was not detected after BMP stimulation. The differential expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction in mesenchymal cells.

Hepatocyte growth factor contributes to fracture repair by upregulating the expression of BMP receptors.

UNLABELLED: Hepatocyte growth factor (HGF) is activated and the expression of BMP receptors (BMPRs) is induced around the fracture site during the early phase of fracture repair. HGF facilitates the expression of BMPRs in mesenchymal cells. This study suggests that HGF contributes to fracture repair by inducing the expression of BMPRs. INTRODUCTION: The precise mechanisms that control the upregulation of BMP, BMPRs, and other molecules involved in bone repair are not completely understood. In this study, we hypothesized that HGF, activated through the action of thrombin on the HGF activator, may enhance BMP action through the local induction of BMP or BMPRs. MATERIALS AND METHODS: Callus samples from tibial fractures in mice were harvested for immunohistochemical analysis of HGF and phosphorylated c-Met, for in situ hybridization of BMPRs, and for real-time RT-PCR analysis for the expression of HGF, c-Met, and BMPRs. To study the changes in gene expression of BMPRs in response to HGF, C3H10T1/2 cells were cultured with or without HGF and harvested for real-time RT-PCR and for Western blot analysis. To evaluate the contribution of HGF to the biological action of BMP2, C3H10T1/2 cells and primary muscle-derived mesenchymal cells were precultured with HGF and cultured with BMP2. In addition, the expression of the luciferase gene linked to the Id1 promoter containing the BMP responsive element and alkaline phosphatase (ALP) activity were assayed. RESULTS: Positive immunostaining of HGF and phosphorylated c-Met was detected around the fracture site at 1 day after the fracture was made. mRNA expression of BMPRs was increased 1 day after fracture and localized in mesenchymal cells at the fracture site. From an in vitro study, the expression of mRNA for BMPRs was elevated by treatment with HGF, but the expression of BMP4 did not change. Western blot analysis also showed the upregulation of BMPR2 by HGF treatment. The results from the luciferase and ALP assays indicated increased responsiveness to BMPs by treating with HGF. CONCLUSIONS: This study indicates that HGF is activated and expressed at the fracture site and that HGF induces the upregulation of BMPRs in mesenchymal cells. Furthermore, HGF may facilitate BMP signaling without altering the expression of BMP molecules.