Members Of Bone Morphogenetic Proteins Research Articles

Assessment of serum levels of bone morphogenetic proteins as sensitive biomarkers for early prediction of acute myocardial infarction.

Although acute myocardial infarction (AMI) is a life-threatening disease, rapid diagnosis may reduce mortality risk. Despite being highly specific for AMI, cardiac troponin I (cTnI) does not distinguish between the etiologically diverse of myocardial injury that may be due to MI or non-MI causes. Bone morphogenetic proteins (BMPs), which are important modulators for cardiac morphogenesis, are members of the transforming growth factor-beta superfamily. This work intended to evaluate the utility of 2 members of BMPs (BMP2 and BMP4) as diagnostic biomarkers for AMI. This study included 110 AMI patients and 30 healthy volunteers. AMI patients were subdivided into 39 patients without hypercholesterolemia or diabetes mellitus (AMI without HC or DM), 33 patients with hypercholesterolemia (AMI with HC), and 38 patients with diabetes mellitus (AMI with DM). BMP2 and BMP4 levels were assessed in all subjects. The results showed that serum levels of both BMPs showed significant elevation in all AMI patients compared to healthy controls. In contrast, serum BMP2 level only was significantly elevated in AMI patients with DM compared to those without HC or DM and therefore was able to discriminate between the two subgroups. Notably, the diagnostic efficacy of the two BMPs was improved when combined. In conclusion, the two BMPs are good diagnostic biomarkers for AMI. Nevertheless, BMP2 had a higher diagnostic performance than BMP4 in discriminating AMI with DM from AMI without HC or DM with AUC 0.919 (p=0.001) and the highest PPV (100%), meanwhile, BMP4’s AUC is 0.891 (p=0.001) and a PPV (85.7%).

Structural Mapping of BMP Conformational Epitopes and Bioengineering Design of Osteogenic Peptides to Specifically Target the Epitope-Binding Sites.

Human bone morphogenetic proteins (BMPs) constitute a large family of cytokines related to members of the transforming growth factor-β superfamily, which fulfill biological functions by specificity binding to their cognate type I (BRI) and type II (BRII) receptors through conformational wrist and linear knuckle epitopes, respectively. We systematically examined the intermolecular recognition and interaction between the BMP proteins and BRI receptor at structural, energetic and dynamic levels. The BRI-binding site consists of three hotspot regions on BMP surface, which totally contribute ~70% potency to the BMP-BRI binding events and represent the core sections of BMP conformational wrist epitope; the contribution increases in the order: hotspot 2 (~8%) < hotspot 3 (~20%) < hotspot 1 (~40%). Multiple sequence alignment and structural superposition revealed a consensus sequence pattern and a similar binding mode of the three hotspots shared by most BMP members, indicating a high conservation of wrist epitope in BMP family. The three hotspots are natively folded into wellstructured U-shaped,, loop and double-stranded conformations in BMP proteins, which, however, would become largely disordered when splitting from the protein context to derive osteogenic peptides in free state, thus largely impairing their rebinding capability to BRI receptor. In this respect, cyclization strategy was employed to constrain hotspot 1/3-derived peptides into a native-like conformation, which was conducted by adding a disulfide bond across the ending arms of linear peptides based on their native conformations. Fluorescence-based assays substantiated that the cyclization can effectively improve the binding affinities of osteogenic peptides to BRI receptor by 3-6-fold. The cyclic peptides also exhibit a good selectivity for BRI over BRII (>5-fold), confirming that they can specifically target the wrist epitope-binding site of BRI receptor. The rationally designed cyclic peptides can be regarded as the promising lead entities that should be further chemically modified to enhance their in vivo biological stability for further bioengineering therapeutic osteogenic peptides againstchondrocyte senescence and bone disorder.

Enhanced BMP signalling causes growth plate cartilage dysrepair in rats

Growth plate cartilage injuries often result in bony repair at the injury site and premature mineralisation at the uninjured region causing bone growth defects, for which underlying mechanisms are unclear. With the prior microarray study showing upregulated bone morphogenetic protein (BMP) signalling during the injury site bony repair and with the known roles of BMP signalling in bone healing and growth plate endochondral ossification, this study used a rat tibial growth plate drill-hole injury model with or without systemic infusion of BMP antagonist noggin to investigate roles of BMP signalling in injury repair responses within the injury site and in the adjacent "uninjured" cartilage. At days 8, 14 and 35 post-injury, increased expression of BMP members and receptors and enhanced BMP signalling (increased levels of phosphorylated (p)-Smad1/5/8) were found during injury site bony repair. After noggin treatment, injury site bony repair at days 8 and 14 was reduced as shown by micro-CT and histological analyses and lower mRNA expression of osteogenesis-related genes Runx2 and osteocalcin (by RT-PCR). At the adjacent uninjured cartilage, the injury caused increases in the hypertrophic zone/proliferative zone height ratio and in mRNA expression of hypertrophy marker collagen-10, but a decrease in chondrogenesis marker Sox9 at days 14 and/or 35, which were accompanied by increased BMP signalling (increased levels of pSmad1/5/8 protein and BMP7, BMPR1a and target gene Dlx5 mRNA). Noggin treatment reduced the hypertrophic zone/proliferative zone height ratio and collagen-10 mRNA expression, but increased collagen-2 mRNA levels at the adjacent growth plate. This study has identified critical roles of BMP signalling in the injury site bony repair and in the hypertrophic degeneration of the adjacent growth plate in a growth plate drill-hole repair model. Moreover, suppressing BMP signalling can potentially attenuate the undesirable bony repair at injury site and suppress the premature hypertrophy but potentially rescue chondrogenesis at the adjacent growth plate.

Bone Morphogenetic Protein (BMP)4 But Not BMP2 Disrupts the Barrier Integrity of Retinal Pigment Epithelia and Induces Their Migration: A Potential Role in Neovascular Age-Related Macular Degeneration.

Disruption of retinal pigment epithelial (RPE) barrier integrity and RPE migration are hallmark features in neovascular age-related macular degeneration (nAMD), but the underlying causes and pathophysiology are not completely well-defined. Herein, we aimed to evaluate the effect of bone morphogenetic proteins (BMPs) on the barrier function and migration of RPE. In particular, we investigated the role of BMP2 and BMP4 in these processes as our analysis of RNA-sequencing (seq) data from human donor eyes demonstrated that they are highly differentially expressed BMP members in macular RPE/choroid versus macular retina. We used electrical cell-substrate impedance sensing (ECIS) system to monitor precisely in real time the barrier integrity and migration of ARPE-19 after treatment with various concentrations of BMP2 or BMP4. Immunofluorescence was also used to assess the changes in the expression and the organization of the key tight junction protein, zona occludens (ZO)-1, in ARPE-19 cells under BMP2 or BMP4 treatment. This was followed by measuring the activity of matrix metalloproteinases (MMPs). Finally, RNA-seq and ELISA were used to determine the local and circulating levels of BMP2 and BMP4 in retinas and serum samples from nAMD donors. Our ECIS results showed that BMP4 but not BMP2 decreased the transcellular electrical resistance (TER) of ARPE-19 and increased their migration in comparison with control (vehicle-treated cells). Furthermore, immunofluorescence showed a disorganization of ZO-1 in BMP4-treated ARPE-19 not in BMP2-treated cells or vehicle-treated controls. This effect of BMP4 was associated with significant increases in the activity of MMPs, specifically MMP2. Lastly, these results were corroborated by additional findings that circulating but not local BMP4 levels were significantly higher in nAMD donor samples compared to controls. Collectively, our results demonstrated unreported effects of BMP4 on inducing RPE dysfunction and suggest that BMP4 but not BMP2 may represent a potential therapeutic target in nAMD.

CLINICAL APPLICATION OF BONE MORPHOGENETIC PROTEIN ON FRACTURE HEALING PROCESS

Delayed union or non-union often occurs in bone healing fracture process. The clinical application of Bone Morphogenetic Proteins (BMPs) has helped an increasing number of patients achieve bone regeneration in clinical area lacking simple solutions for difficult bone healing situations. Many researches has proved that BMPs improved fracture healing regeneration. In this review article, introduction and current clinical issues are summarized on efficacy and function of BMPs. Currently, rhBMP-2 and rhBMP-7 have shown clinical significance but other subfamily members of BMPs still lacked of evidence.

Deletion of Lrp4 increases the incidence of microphthalmia

Microphthalmia is a malformation that reduces the size of the ocular globe. The etiologies of this anomaly are various, but the genetic background appears to have a predominant influence on its development through mutations of genes controlling ocular developmental processes. LRP4 is a type I single transmembrane protein that is essential for the formation of neuromuscular junctions. We created and experimented on homozygous Lrp4-deficient mice and found the microphthalmia phenotype in their eyes. The loss of Lrp4 resulted in an elevated incidence of microphthalmia and affected the mRNA expression of the members of bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, and WNT signaling pathways and of several pathogenic genes for microphthalmia. Moreover, the loss of Lrp4 enhanced the incidence of aberrant retinal folds, which appeared pleated and corrugated in the eyeball.

Disparate phospho-Smad2 levels in advanced type 2 diabetes patients with diabetic nephropathy and early experimental db/db mouse model

Uncontrolled activation of transforming growth factor beta (TGF-β) family members is hypothesized to participate in type 2 diabetes (T2D) dependent diabetic nephropathy (DN). We evaluated and compared downstream activation of the Smad2-signaling pathway in kidney samples from T2D patients to kidneys from the T2D model of leptin receptor deficient db/db mouse. Furthermore, expression of TGF-β family members was evaluated to elucidate molecular mechanisms in the mouse model. Kidney samples from patients with advanced stages of DN showed elevated pSmad2 staining whereas db/db mouse kidneys surprisingly showed a decrease in pSmad2 in the tubular compartment. Structurally, kidney tissue showed dilated tubules and expanded glomeruli, but no clear fibrotic pattern was found in the diabetic mice. Selective TGF-β family members were up-regulated at the mRNA level. Antagonists of bone morphogenetic protein (BMP) ligands, such as Gremlin1, USAG1 and Sclerostin, were strongly up-regulated suggesting a dampening effect on BMP pathways. Together, these results indicate a lack of translation from T2D patient kidneys to the db/db model with regards to Smad signaling pathway. It is plausible that a strong up-regulation of BMP antagonizing factors account for the lack of Smad1/5/8 activation, in spite of increased expression of several BMP members.

Bone morphogenetic proteins: Relationship between molecular structure and their osteogenic activity

Bone morphogenetic proteins (BMPs) are a family of potent, multifunctional growth factors belonging to transforming growth factor-β (TGF-β). They are highly conservative in structures. Over 20 members of BMPs with varying functions such as embryogenesis, skeletal formation, hematopoiesis and neurogenesis have been identified in human body. BMPs are unique growth factors that can induce the formation of bone tissue individually. BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes. BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic (Smads) and mitogen activated protein kinase (MAPK) pathways. It has been demonstrated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 play an important role in bone formation. This article focuses on the molecular characterization of BMPs family members, mechanism of osteogenesis promotion, related signal pathways of osteogenic function, relationships between structure and osteogenetic activity, and the interactions among family members at bone formation.

Structural and morphometric comparison of the molar teeth in pre-eruptive developmental stage of PACAP-deficient and wild-type mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain

BackgroundBoth bone morphogenetic proteins (BMPs) and histone deacetylases (HDACs) have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling.ResultsUsing neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level.ConclusionsSTAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.

The signaling and functions of heterodimeric bone morphogenetic proteins

Heterodimeric bone morphogenetic proteins (BMPs) consist of disulfide-linked dimeric monomers derived from different BMP members. Owing to this specific constitution pattern, they bear high affinity to both type I and type II BMP receptors simultaneously. Meanwhile, the antagonism efficiency of extracellular antagonists to heterodimeric BMPs is also significantly lower than that to homodimeric ones. All these specific properties confer heterodimeric BMPs with distinct signaling and bio-functions that are characterized by more speediness, lower concentration/dose threshold and higher efficiency than homodimeric BMPs. Consequently, heterodimeric BMPs bear promising application potential in inducing osteogenesis. In addition, they may play indispensible roles in organogenesis. In this review, we summarize the current knowledge of heterodimeric BMPs in their signaling pathways and bio-functions.

Differential regulation of gonadotropin receptors by bone morphogenetic proteins in the zebrafish ovary

Follicle-stimulating hormone receptor (fshr) and luteinizing hormone/choriogonadotropin receptor (lhcgr) exhibit differential temporal expression patterns during zebrafish folliculogenesis with fshr being dominant during vitellogenic growth and lhcgr increasing its expression dramatically before maturation. The dynamic and distinct expression patterns of fshr and lhcgr suggest that they are under tight regulatory control. However, the underlying mechanisms for the differential expression of the two receptors remain unknown. We have recently demonstrated that members of bone morphogenetic protein (BMP) family are largely expressed in the oocyte, while their receptors are exclusively localized on the follicle cells, suggesting a potential paracrine signaling from the oocyte to the follicle cells by BMPs. In this study, we investigated the effects of zebrafish BMP2b (zfBmp2b) and BMP4 (zfBmp4) on the expression of fshr and lhcgr using a novel co-culture approach. The recombinant zfBmp2b or zfBmp4-producing CHO cells were co-cultured with the zebrafish follicle cells followed by real-time qPCR analysis of fshr and lhcgr expression. Our results showed that zfBmp2b and zfBmp4 both down-regulated fshr, while up-regulated lhcgr expression at 24h of co-culturing. This finding, together with the high expression level of BMP receptors in the follicle cells prior to oocyte maturation, strongly suggests a potential role for BMPs in the differential expression of fshr and lhcgr, especially in the full-grown follicles before maturation. As BMPs are largely expressed in the oocyte, this also implies an important role for the oocyte in orchestrating the differentiation and function of the follicle cells.

Bone Morphogenetic Proteins Inhibit CD40L- and IL-21-Induced Ig Production In Human B Cells: Differential Effects of BMP-6 and BMP-7

AbstractAbstract 1745 Introduction:TGF-β has been shown to play an important role in directing class switch recombination in human B cells. Bone morphogenetic proteins (BMPs) are members of the TGF-β family, and we have previously shown that BMP-6 inhibits proliferation of B cell progenitors as well as mature B cells in humans. However, how various BMP members affect Ig production and B-cell maturation to plasma cells have not been investigated. Here we studied the effects of various BMPs in CD27- naive and CD27+ memory B cells from peripheral blood of healthy human donors. Methods:We used flow immunomagnetic beads and flow cytometry cell sorting to purify CD19+CD27- naive and CD19+CD27+ memory B cells from peripheral blood of healthy donors, and investigated BMP induced effects on in vitro proliferation, cell death and Ig production as measured by thymidine incorporation assay, propidium iodide staining and ELISA, respectively. To separate direct inhibition of plasma cell differentiation from indirect effects via suppression of proliferation and induction of apoptosis, CFSE tracking of cell division was combined with immunophenotyping of cultured cells to identify CD27+CD38+ plasma cells. Expression of transcription factors and AICDA were measured by quantitative real-time PCR. Results:BMP-2, -4, -6 and -7 specifically inhibited CD40L- and IL-21-induced production of IgM, IgG and IgA. BMP-6 was the most potent inhibitor, reducing the Ig production by 70% in memory B cells and more than 50% in naive B cells. By investigating the mechanisms for reduced Ig production, we found a striking difference between the structurally similar BMP-6 and BMP-7. BMP-6 directly inhibited differentiation to CD27+CD38+ plasma cells, whereas BMP-7 only had minor effects on differentiation. Instead, BMP-7 mainly affected Ig production indirectly by inducing apoptosis. Furthermore, we explored BMP-6-induced signaling and gene regulation in more detail in memory B cells. BMP-6 up-regulated Id1, Id2 and Id3 gene expression in CD40L- and IL-21-stimulated cells (6.1-fold, 1.7-fold and 4.0-fold induction after 48 hours culture with BMP-6, respectively). In contrast, BMP-6 potently inhibited CD40L- and IL-21-induced upregulation of the transcription factor XBP-1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected, indicating that BMP-6 only modulates late events of plasma cell development. Conclusion:These results show that BMPs potently suppress Ig production in mature human B cells by inhibiting differentiation or by inducing cell death. Disclosures:No relevant conflicts of interest to declare.

Bone morphogenetic protein‐7 regulates differentially the mRNA expression of bone morphogenetic proteins and their receptors in rat achilles and patellar tendon cell cultures

Previous animal studies have suggested that certain bone morphogenetic proteins (BMPs) may be useful therapeutically in treating tendon healing. To better understand the relationship among the different BMPs in the healing process, we initiated the present study to examine the effects of a member of the BMP family, BMP-7 (also called Osteogenic Protein-1) on the temporal and spatial expression patterns of other BMPs and the BMP receptors in cell cultures of adult rat Achilles and Patellar tendons. Cultures from both tendon types expressed detectable but variable levels of biochemical markers characteristics of tendons. RNAs coding for type II collagen and transcription factors Six1, Scleraxis, and Tendin were detected in both types of cultures. Distinct patterns of expression of several BMP members and their receptors were observed in these cultured cells and BMP-7 exerted differential effects on their expression. The findings may have implications in the treatment of different tendon injuries with BMPs.

Bone Morphogenetic Protein-9 Is a Circulating Vascular Quiescence Factor

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

Expression of the Bone Morphogenetic Protein Receptor 1A Gene (BMPR1A) in Adipose Tissue and BMPR1A Genetic Variation are Associated with Human Obesity

Family of bone morphogenetic proteins (BMP) is involved in regulation of adipogenesis. We measured the mRNA expression of BMP members in paired samples of visceral and subcutaneous adipose tissue from 198 subjects and examined its relation to obesity. Amongst others, mRNA expression of the human bone morphogenetic protein receptor 1A (BMPR1A) was significantly increased in both visceral and subcutaneous adipose tissue of 51 overweight (BMI>25 and <30kg/m2) and 91 obese (BMI>30kg/m2) subjects compared with 56 lean subjects (BMI<25kg/m2) (P<0.05 and P<0.001, respectively). BMPR1A is one of the three specific BMP-receptors which are essential for the bone morphogenetic protein signalling. The BMPR1A gene maps to human chromosome 10q23 and spans about 168 kb. To determine whether genetic variants within the BMPR1A are associated with mRNA expression in fat and whether these variants affect the pathophysiology of human obesity and type 2 diabetes (T2D), we sequenced the BMPR1A (13 exons, exon-intron boundaries, 5′ and 3′ UTRs) in DNA samples from 48 non-related Caucasian subjects to identify genetic variants. Twenty representative variants including HapMap tagging SNPs (single nucleotide polymorphisms) (www.hapmap.org) were genotyped for subsequent association studies on quantitative traits in 1808 German Caucasians with detailed metabolic testing (896 subjects without T2D and 912 subjects with T2D). In a case control study including 461 lean (BMI<25kg/m2) and 678 obese (BMI>30kg/m2) subjects, four SNPs (rs7095025, rs7922846, rs11202222 and rs10788528) were significantly associated with obesity (all P<0.05, adjusted for age and sex). Consistent with this, we found significant associations between BMPR1A SNPs and obesity/diabetes related quantitative traits (body mass index (BMI), circulating serum leptin, fasting plasma glucose, 2-hr plasma glucose in oral glucose tolerance test; all P<0.05, adjusted for age, sex and BMI) in 896 subjects without T2D. Finally, the rs17426348 was moderately associated with T2D in 912 cases with T2D and 718 healthy controls with normal glucose tolerance (P=0.05, adjusted for age, sex and BMI). In conclusion, association of both, BMPR1A expression in fat and BMPR1A genetic variation with obesity suggest that this gene may play a role in the pathophysiology of human obesity and T2D.

Knockdown of the Intraflagellar Transport Protein IFT46 Stimulates Selective Gene Expression in Mouse Chondrocytes and Affects Early Development in Zebrafish

Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

Fracture repair and bone morphogenetic protein (BMP)

The process of fracture healing involves a number of regenerative mechanisms underlying the skeletal systems, and bone morphogenetic protein (BMP) has been believed to be a key molecule during the reaction. Recent investigations showed that several members of BMPs are induced and activated by the impact of fracture, and play important roles via BMP receptors/Smads pathways. BMPs are newly synthesized by callus-forming cells near the fracture site, and form a "BMP-network" at the fracture callus. The "BMP-network" contributes to the regulation of callus formation, and the network contains noggin, sonic hedgehog (Shh), hepatocyte growth factor (HGF), and vascular endothelial cell growth factor (VEGF). Potential roles of BMPs during fracture repair open the way towards the formulation of new therapeutic strategies for promising fracture management. Recent molecular technologies with use of recombinant BMP and gene therapy will lead to the development of less-invasive and more successful fracture treatment.

Transcriptional mRNA of BMP-2, 3, 4 and 5 in trigeminal nerve, benign and malignant peripheral nerve sheath tumors.

The aim of our study was to document whether relationships existed among bone morphogenetic proteins (BMPs), peripheral nerve and neoplastic lesions of nerve sheath tumors. The mRNA transcriptions of BMP-2, 3, 4 and 5 in 10 cases of schwannoma, three cases of malignant schwannoma and two cases of trigeminal neuralgia were detected using an in situ hybridization technique. Our results demonstrated that the myelin sheaths of Schwann cell from the peripheral neuroectomy of trigeminal neuralgia positively expressed mRNA of BMP-2, 3, 4, and 5. The most interesting finding was that the nerve fibers of trigeminal nerve showed only BMP-2 positive staining. All of the neoplastic lesions of nerve sheath showed a consistent but variant expression of BMP-2, 3, 4, and 5. The expression signals of BMP-2, 3, 5 mRNA in malignant schwannoma were relatively lower than in benign lesions except for the expression of BMP-4 mRNA. Our results indicated that selected members of BMPs were expressed in the peripheral nerves that might contribute to the health maintenance, proliferation, regeneration and neoplastic transformation of the peripheral nerve system. Furthermore, the effects of BMP-2, 3, 4 and 5 on peripheral nervous system during neoplastic transformation might be widespread, diverse and antagonistic.

Distribution of genes for bone morphogenetic protein-4, -6, growth differentiation factor-5, and bone morphogenetic protein receptors in the process of experimental spondylosis in mice.

Because little is known about the molecular mechanisms underlying the process of spondylosis, the authors examined the extent of genetic localization of several members of bone morphogenetic protein (BMP) and BMP receptors in chondrogenesis during the process of inducing spondylosis in their previously established experimental mice model. Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spine was harvested chronologically, and histological sections were prepared. Messenger RNA for BMP-4, growth and differentiation (GDF)-5, BMP-6, and BMP receptors (ALK-3, -6, and BMP-RII) was localized in the tissue sections by in situ hybridization. In the early stage, BMP-4-derived mRNA was localized mainly in cells in the anterior margin of the cervical discs, together with ALK-6 and BMP-RII mRNA. No GDF-5 and BMP-6 mRNA was detected at this stage. In the late stage, cells positive for BMP-4 decreased, whereas GDF-5 and BMP-6 mRNA were localized in cells undergoing chondrogenesis. The ALK-3 mRNA began to appear in this stage, as did ALK-6 and BMP-RII. The localization of transcripts for BMP-4, -6, and GDF-5 as well as BMP receptors shown during the present experimental model indicate the possible involvement of molecular signaling by these BMPs in the chondrogenic progress in spondylosis.