Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.

Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Roles of Bone Morphogenetic Protein Type I Receptors and Smad Proteins in Osteoblast and Chondroblast Differentiation

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

Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

International audience

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Bovine Bone Matrix Action Associated With Morphogenetic Protein in Bone Defects in Rats Submitted to Alcoholism

Extended excessive alcohol use causes changes in bone tissue, thus affecting osteogenesis. The objective of this study was to evaluate if demineralized bone matrix (Gen-ox (R)) associated with bone morphogenetic protein (Gen-pro (R)) changes bone neoformation in rats submitted to experimental alcoholism. Forty male rats (Rattus norvegicus) were separated into 2 groups of 20 animals each: Group E1, which received ethyl alcohol at 25% and had the surgical cavity filled in only with blood clot; and Group E2. which received ethyl alcohol at 25% and had the surgical cavity filled in with demineralized bovine cortical bone associated with bone morphogenetic protein. The animals were submitted to a three-week period of gradual adaptation to alcohol, and then continued receiving alcohol at 25% for 90 days, when the surgical cavity was made. After the surgery, the animals continued consuming alcohol until reaching the sacrifice periods of 10, 20, 40, and 60 days, when the tibias were removed for histological processing. Results showed that surgical cavity repair and bone marrow reorganization occurred faster in Group E1 than in Group E2. At the end of the experiment, it was observed that animals in Group E2 had thick bony trabeculae surrounding the implanted material particles and a small area of connective tissue in the surface region. In conclusion, the implanted material did not accelerate bone neoformation, rather it served as a structure for osteogenesis.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. (E-mail: a.renno@unifesp.br) (C) 2010 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology.

Protein profile of the luteal phase endometrium by tissue microarray assessment

To investigate the luteal phase endometrial expression of leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), progesterone receptor (PR), claudin 4 (CLDN4), vascular-endothelial growth factor receptor 3 (VEGFR-3), bone morphogenetic protein 4 (BMP-4) and citokeratin 7 (CK-7), we obtained luteal phase endometrial samples from 52 women. Samples were dated and integrated using a tissue microarray (TMA). Samples were immunostained for LIF, IGF-1, PR, CLDN4, VEGFR-3, BMP-4 and CK-7. Frequencies of positive expressions at the early, mid and late luteal phases were compared by two proportions test. Concomitant expression of these proteins was assessed with Chi-square or Fischer`s test. The frequency of LIF was positively correlated to the frequency of IGF-1 (r = 0.99; p < 0.05) and PR (r = 0.99; p < 0.05), and the correlation between IGF-1 and PR tended to be significant (r = 0.98; p < 0.1). The expression of PR was associated with the absence of CLDN4 (p < 0.001). Thus, expression of LIF, IGF-1 and PR are correlated during the luteal phase, and immunohistochemistry for these proteins might be used to assist in the assessment of endometrial maturation. In addition, the expression of CLDN4 and PR was not concomitant, warranting further investigation on the relationship of their endometrial expression.