Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice.

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

A 190 base pair, TGF-ß responsive tooth and fin enhancer is required for stickleback Bmp6 expression.

The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190bp enhancer ~2.5 kilobases 5' of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFß effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFß signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.

The effects of Gremlin1 on human umbilical cord blood hematopoietic progenitors.

Bone morphogenetic proteins (BMPs) support malignant hematopoiesis in CML. Conversely, the multi-functional BMP antagonist Gremlin1 supports self-renewing cancer stem cells of other malignancies. Inhibition of BMP signaling in CML, or of Gremlin1 in solid tumors, may therefore have therapeutic potential. However, since BMPs regulate hematopoietic stem cell (HSC) decisions in the stem cell niche, it is necessary to determine how Gremlin1 influences normal HSC. We examined the effects of Gremlin1 on long-term culture-initiating cells (LTC-IC) and transplantable hematopoietic stem cells (SCID-repopulating cells: SRC) in human umbilical cord blood. Gremlin1 inhibited BMP signaling, downregulated BMP-6 and cyclin E2 expression and upregulated hairy and enhancer of split-1 (HES-1; a Notch transcriptional target) and Hedgehog interacting protein-1 (HHIP-1; an inhibitor of Hedgehog signaling). The functional effects of Gremlin1 on SRC, i.e. skewing of their myelopoietic:lymphopoietic potential towards B lymphopoiesis without affecting long-term engraftment potential, were entirely consistent with changes in gene expression induced by Gremlin1. Since both BMPs and Gremlin1 are secreted by osteoblasts in vivo, our studies provide potential insights into the molecular regulation of hematopoiesis in the stem cell niche. These results also suggest that Gremlin1 (and possibly its mimetics that may be developed for therapeutic use) may not adversely affect normal human hematopoietic stem cell survival, though they may reduce their myelopoietic potential.

Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP-6 induction.

BACKGROUND: Androgen ablation is the first-line therapy for patients with metastatic prostate cancer (CaP). However, castration resistance will eventually emerge. In the present study, we have investigated the role of bone morphogenetic protein-6 (BMP-6) in the development of castration-resistant prostate cancer (CRPC) in the context of bone metastases. METHODS: We initially investigated the clinical course of 158 men with advanced CaP who were treated with primary androgen deprivation therapy. To elucidate the underlying mechanism of CRPC in the context of bone metastases, we examined the impact of bone stromal cells on CaP in the absence of androgens using a co-culture model. RESULTS: In the 158 patients, we found that the median time to prostate-specific antigen progression was significantly shorter when bone metastases were present (14 months (95% CI, 10.2-17.8 months) vs 57 months (95% CI, 19.4-94.6 months)). These results suggest that bone-tumour interactions may accelerate castration resistance. Consistent with this hypothesis, in vitro co-cultures demonstrated that CaP cells proliferated under an androgen-depleted condition when incubated with bone stromal cells. Mechanistically, gene expression analysis using quantitative polymerase chain reaction arrays showed a dramatic induction of BMP-6 by CaP cell lines in the presence of bone stromal cells. Further studies revealed that WNT5A derived from bone stromal cells induced the expression of BMP-6 by CaP cells; BMP-6 in turn stimulated cellular proliferation of CaP cells in an androgen-deprived media via a physical interaction between Smad5 and ß-catenin. Intracellularly, WNT5A increased BMP-6 expression via protein kinase C/NF-κB pathway in CaP cell lines. CONCLUSIONS: These observations suggest that bone-CaP interaction leads to castration resistance via WNT5A/BMP-6 loop.

Expression of bone morphogenetic protein-6 in dental follicle stem cells and its effect on osteogenic differentiation.

The dental follicle (DF) plays an essential role in tooth eruption via regulation of bone resorption and bone formation. Bone morphogenetic protein-6 (BMP6) expression in the DF is coincident with bone growth in the tooth crypt. DF stem cells (DFSCs) have been shown to possess strong osteogenic capability. This study aims to determine the expression of BMP6 in DFSCs and to elucidate the role of BMP6 in the osteogenesis of DFSCs. DFSCs and their non-stem cell counterpart, DF cells (DFCs), were obtained from the DFs of rat pups. We showed that expression of BMP6 was significantly higher in the DFSCs than in the DFCs. DFSCs lost osteogenic capability during in vitro expansion, and DFSCs in late passages had reduced BMP6 expression as compared to early passages of DFSCs when they were subjected to osteogenic induction. Addition of exogenous human recombinant BMP6 (hrBMP6) to the osteogenic medium dramatically enhanced the osteogenesis of the late-passage DFSCs. Knockdown of BMP6 by short interfering RNA in the DFSCs in early passages resulted in a decrease in osteogenesis, which could be restored by addition of hrBMP6. We concluded that DFSCs need to express high levels of BMP6 to maintain their osteogenesis capability. Increased BMP6 expression seen in vivo in the DF may reflect the activation of DFSCs for osteogenic differentiation for bone growth during tooth eruption.

Microarray analysis of gene expression of bone marrow stem cells cocultured with salivary acinar cells.

BACKGROUND/PURPOSE: Our previous work has demonstrated that rat bone marrow stem cells (BMSCs) can transdifferentiate into α-amylase-producing cells after coculture with rat submandibular gland acinar cells. These transdifferentiated cells may be used for regeneration of damaged salivary gland. The purpose of this study was to investigate the global gene expression of rat BMSCs cocultured with rat submandibular gland acinar cells and the factors inducing this transdifferentiation. METHODS: Rat BMSCs were indirectly cocultured with rat submandibular gland acinar cells by using the double chamber system for 5 and 10 days. The global gene expression of BMSCs during transdifferentiation into acinar cells was investigated by microarray analysis. RESULTS: A total of 45,018 probes were used and 41,012 genes were detected. After coculture for 5 days, 1409 genes were upregulated more than twofold and 1417 genes were downregulated more than twofold (p<0.005). Moreover, after coculture for 10 days, 1356 genes were upregulated more than twofold and 1231 genes were downregulated more than twofold (p<0.005). Bone morphogenetic protein (BMP)-6 was one of the top-ranked upregulated genes. The hub genes were interleukin-6 and CCAAT/enhancer-binding protein ß (CEBPB) in the early and late response gene groups, respectively. CONCLUSION: This is believed to be the first study on the global gene expression of rat BMSCs cocultured with rat acinar cells. Many genes related to the function of salivary acinar cells such as those responsible for the production of α-amylase protein were upregulated and many genes related to the differentiation of BMSCs into adipocytes and osteoblasts were downregulated. In addition, BMP-6 gene was found to be highly upregulated. We proposed that three target genes, BMP-6, interleukin-6 and CEBPB, play important roles in the transdifferentiation of BMSCs into acinar cells, and are worthy of further investigation.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

CREBZF, a novel Smad8-binding protein.

Smads are the secondary messengers of the transforming growth factor-ß (TGF-ß) signaling pathway. TGF-ß receptors phosphorylate the Receptor Smads (R-Smads) upon ligand binding; activated R-Smads translocate to the nucleus and function as transcription factors. Among the R-Smads, Smads 1, 5, and 8 mainly mediate signals in the bone morphogenetic proteins (BMPs) pathways, while Smads 2/3 mediate TGF-ß signaling. The regulation of Smads in the TGF-ß signal pathway has been well defined, but the relationship of Smads 1, 5, and 8 to the BMP pathways has been relatively understudied. To understand the specific regulation of BMP mediating Smads, we performed yeast two-hybrid screening using the Mad homology 2(MH2) domain of Smad8 as bait. In this screening, novel Smad-binding protein, CREBZF-a basic region-leucine zipper (bZIP) transcription factor-was identified. The interaction of CREBZF and Smads 1, 5, and 8 was confirmed by immunoprecipitation in a human prostate cancer cell line. Overexpression of CREBZF inhibited the promoter activity of BMP response element and abolished the cell growth inhibition induced by BMP-6. Thus, CREBZF inhibits the function of BMP-6 by interacting with Smads. The identification of this novel Smads-binding protein, among others will help us understand the modulation of BMP-signaling pathways.

Co-expression of bone morphogenetic protein 6 with estrogen receptor a in endometriosis.

BACKGROUND: Bone morphogenetic protein 6 (BMP-6) has decisive role in controlling multiple organogenetic processes, as well as modulating cell differentiation and proliferation. Considering those pleiotropic effects, we focused on determining expression of that multifunctional growth factor in ectopic endometriotic tissues. MATERIALS AND METHODS: In this prospective study, 85 consecutive women with endometriosis were included. All patients underwent gynecological operations due to endometriosis associated problems and tissue specimens were collected from ectopic endometriotic lesions. Immunohistochemical staining of paraffin sections for both BMP-6 and estrogen receptors a (ERa) was performed in all 85 cases using an avidin-biotin-peroxidase procedure. RESULTS: Ectopic endometrium showed intense cytoplastic immunoreactivity to BMP-6 in both epithelium and stroma. In addition, we have demonstrated that BMP-6 expression is highly associated with strong expression of ERa. DISCUSSION: The availability of BMP-6 in the ectopic endometrium may be at least partly involved in the mechanisms of attachment, survival and expansion of endometriosis. Moreover, the statistically significant correlation in expression of BMP-6 and ERa demonstrated in this study may be associated with the development of rich in estrogen microenvironment, but requires further investigation. In conclusion, this is the first study in our knowledge demonstrating strong expression of BMP-6 in endometriosis.

Increased BMP6 levels in the brains of Alzheimer's disease patients and APP transgenic mice are accompanied by impaired neurogenesis.

During aging and in the progression of Alzheimer's disease (AD), synaptic plasticity and neuronal integrity are disturbed. In addition to the alterations in plasticity in mature neurons, the neurodegenerative process in AD has been shown to be accompanied by alterations in neurogenesis. Members of the bone morphogenetic protein (BMP) family of growth factors have been implicated as important regulators of neurogenesis and neuronal cell fate determination during development; however, their role in adult neurogenesis and in AD is less clear. We show here by qRT-PCR analysis that BMP6 mRNA levels were significantly increased in the hippocampus of human patients with AD and in APP transgenic mice compared to controls. Immunoblot and immunohistochemical analyses confirmed that BMP6 protein levels were increased in human AD brains and APP transgenic mouse brains compared to controls and accumulated around hippocampal plaques. The increased levels of BMP6 were accompanied by defects in hippocampal neurogenesis in AD patients and APP transgenic mice. In support of a role for BMP6 in defective neurogenesis in AD, we show in an in vitro model of adult neurogenesis that treatment with amyloid-ß(1-42) protein (Aß) resulted in increased expression of BMP6, and that exposure to recombinant BMP6 resulted in reduced proliferation with no toxic effects. Together, these results suggest that Aß-associated increases in BMP6 expression in AD may have deleterious effects on neurogenesis in the hippocampus, and therapeutic approaches could focus on normalization of BMP6 levels to protect against AD-related neurogenic deficits.

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.

Emerging ideas: treatment of precollapse osteonecrosis using stem cells and growth factors.

BACKGROUND: Osteonecrosis (ON) of the femoral head is a devastating disease affecting young patients at their most productive age, causing major socioeconomic burdens. ON is associated with various etiologic factors, and the pathogenesis of the disease is unknown. Most investigators believe the disease is the result of secondary microvascular compromise with subsequent bone and marrow cell death and defective bone repair. QUESTIONS/HYPOTHESES: We hypothesize that local delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6), which induces angiogenesis and osteogenesis respectively, will reverse the disease process and provide a treatment for precollapse ON. METHOD OF STUDY: We will use genetically engineered bone marrow stem cells, carrying VEGF and BMP-6 genes, to enhance angiogenesis and osteogenesis in necrotic bone of an animal model, by local delivery of growth factor in addition to the bone-forming property of the stem cells. The participation, localization, and fate of the stem cells in the repair process will be evaluated by tracing marker-gene product. Osteogenesis and angiogenesis will be assessed using high-resolution xray CT and immunohistomorphometry quantitatively. Mechanical properties of the repair tissue will be determined using an indentation test of the femoral head. SIGNIFICANCE: We envision that a deliverable or injectable bone graft substitute containing engineered stem cells and therapeutic growth factors will be developed through this proposed study and will provide a much needed treatment for ON.

Bone morphogenetic protein 6 expression in cumulus cells is negatively associated with oocyte maturation.

Bone morphogenetic protein 6 (BMP6) is a regulatory peptide secreted by oocytes and granulosa cells that locally regulates folliculogenesis and follicular development. To determine BMP6 location, we studied BMP6 expression in human follicles using immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and western blot analysis. RT-PCR was performed on 354 individual cumulus cell (CC) masses from 48 women to investigate the relationship between BMP6 mRNA expression in CCs and oocyte developmental potential. Results showed that BMP6 protein was mainly located in oocytes from preantral follicles and in granulosa cells from antral follicles. BMP6 mRNA expression was much higher in oocytes than in CCs and mural granulosa cells (mGCs) from preovulatory follicles (p < 0.01), and BMP6 protein level was higher in CCs than in mGCs (p < 0.05). BMP6 mRNA expression was higher in CCs from immature oocytes than in those from mature oocytes (p < 0.05). However, BMP6 mRNA expression in CCs was not associated with oocyte fertilization, embryo morphological grading, or implantation. In conclusion, BMP6 was mainly expressed in oocytes at all human follicular developmental stages and BMP6 mRNA expression in CCs may be negatively correlated with oocyte maturation. BMP6 expression could therefore be used as a biomarker of oocyte maturation.

Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice.

Transcription of the hepcidin (Hamp) gene is controlled by iron stores and the rate of erythropoiesis. Functional hierarchy between these two stimuli has not yet been completely established. It is also not known whether the erythropoiesis-related downregulation of Hamp expression utilises the bone morphogenetic protein/hemojuvelin (Bmp/Hjv) pathway. Hemojuvelin-mutant (Hjv-/-) mice treated with erythropoietin (EPO) at 50IU/mouse/day for three days displayed marked decrease in Hamp mRNA, demonstrating that hemojuvelin is not an indispensable component in EPO-induced Hamp gene downregulation. Irradiation of Hjv-/- mice prevented the EPO-induced decrease of Hamp mRNA, highlighting the role of erythropoiesis in Hamp gene regulation by EPO. After a single injection of EPO, Hamp mRNA levels were not significantly changed at 6h, but decreased at 10 and 24h. Chronic bleeding decreased hepatic Bmp6 mRNA levels; however, repeated EPO treatment did not change Bmp6 mRNA, suggesting that the erythropoietic regulator(s) act independently of the Bmp/Hjv pathway. Pretreatment of C57BL/6 mice with iron (5mg/mouse) almost completely inhibited the EPO-induced decrease of Hamp mRNA. This result suggests that administration of EPO to patients with transfusional iron overload is probably not associated with the risk of additional absorption of substantial amounts of iron from the diet.

Triclosan inhibition of acute and chronic inflammatory gene pathways.

AIM: We sought to determine whether triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether), an extensively used anti-plaque agent with broad-spectrum anti-microbial activity, with reported anti-inflammatory effects via inhibition of prostaglandin E2 and interleukin 1 (IL-1)beta, could also more broadly suppress multiple inflammatory gene pathways responsible for the pathogenesis of gingivitis and periodontitis. MATERIALS AND METHODS: As an exploratory study, the effects of triclosan on the inflammatory gene expression profile were assessed ex vivo using peripheral whole blood samples from eight periodontally healthy donors. Ten-millilitres whole blood aliquots were incubated 2 h with 0.3 microg/ml Escherichia coli lipopolysaccharide (LPS) with or without 0.5 microg/ml triclosan. Affymetrix microarray gene expression profiles from isolated leucocytes and pathway-specific quantitative polymerase chain reaction arrays were used to investigate changes in expression of target cytokines and cell signalling molecules. RESULTS: Ex vivo human whole blood assays indicated that triclosan significantly down-regulated the LPS-stimulated expression of Toll-like receptor signalling molecules and other multiple inflammatory molecules including IL-1 and IL-6 and the dampening of signals that activate the T-helper type 1 acquired immune response via suppression of CD70 with concomitant up-regulation of growth factors related to bone morphogenetic protein (BMP)2 and BMP6 synthesis. CONCLUSIONS: Anti-inflammatory effects were found in this exploratory survey, including suppression of microbial-pathogen recognition pathway molecules and the suppression of acute and chronic mediators of inflammation.

BMPs in periprosthetic tissues around aseptically loosened total hip implants.

BACKGROUND AND PURPOSE: Primary and dynamically maintained periprosthetic bone formation is essential for osseointegration of hip implants to host bone. Bone morphogenetic proteins (BMPs) play a role in osteoinductive bone formation. We hypothesized that there is an increased local synthesis of BMPs in the synovial membrane-like interface around aseptically loosened total hip replacement (THR) implants, as body attempts to generate or maintain implant fixation. PATIENTS AND METHODS: We compared synovial membrane-like interface tissue from revised total hip replacements (rTHR, n = 9) to osteoarthritic control synovial membrane samples (OA, n = 11. Avidin-biotin-peroxidase complex staining and grading of BMP-2, BMP-4, BMP-6, and BMP-7 was done. Immunofluorescence staining was used to study BMP proteins produced by mesenchymal stromal/stem cells (MSCs) and osteoblasts. RESULTS AND INTERPRETATION: All BMPs studied were present in the synovial lining or lining-like layer, fibroblast-like stromal cells, interstitial macrophage-like cells, and endothelial cells. In OA and rTHR samples, BMP-6 positivity in cells, inducible by the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta, predominated over expression of other BMPs. Macrophage-like cells positive for BMP-4, inducible in macrophages by stimulation with particles, were more frequent around loosened implants than in control OA samples, but apparently not enough to prevent loosening. MSCs contained BMP-2, BMP-4, BMP-6, and BMP-7, but this staining diminished during osteogenesis, suggesting that BMPs are produced by progenitor cells in particular, probably for storage in the bone matrix.

The prognostic significance of BMP-6 signaling in prostate cancer.

The importance of bone-morphogenetic proteins in prostate cancer is well recognized. Bone-morphogenetic protein-6 overexpression has been shown to increase the aggressiveness and invasiveness of prostate cancer cells. Recent studies on noggin and sclerostin, potent inhibitors of bone-morphogenetic protein signaling, have found that noggin also modifies the ability of prostate cancer cells to metastasize to bone. Taken together, these results suggest that bone-morphogenetic protein-6 signaling is important in prostate cancer progression. Our study investigated the expression of bone-morphogenetic protein-6, noggin and sclerostin in human prostate specimens (n=136) by immunohistochemical staining. We found that bone-morphogenetic protein-6 was increased (P<0.001), whereas sclerostin was decreased (P=0.004) in prostate cancer compared with nodular hyperplasia. In addition, significantly higher level of bone-morphogenetic protein-6 expression was observed in high-grade prostate cancer with Gleason score >or=7 (P=0.027). Bone-morphogenetic protein-6, noggin and sclerostin alone could not predict the development of distant metastasis in our patient cohort. However, high level of bone-morphogenetic protein-6 and low level of noggin, or high level of bone-morphogenetic protein-6 and low level of both noggin and sclerostin expression in primary prostate cancer significantly predicted development of distant metastasis. The predictive value was still valid when only high-grade prostate cancers were included or when patients with secondary lesion other than bone were excluded. Taken together, these results suggest that a high level of bone-morphogenetic protein-6 signaling, resulting from increased expression of bone-morphogenetic protein-6 and decreased expression of its inhibitors, might promote the development of prostate cancer metastases. Our results also imply the potential use of bone-morphogenetic protein-6, noggin and sclerostin expression together as a prognostic predictor for metastatic progression of prostate cancer.

BMP signaling pathways affect differently migration and invasion of esophageal squamous cancer cells.

Bone morphogenetic proteins (BMPs) are broadly involved in normal embryo development and abnormal pathological process such as cancer. The complexity and diversity of BMPs and their signaling pathways impose quite different or even conflicting effects on clinical traits of tumors. The aim of the present study was to investigate whether different BMPs, including BMP2, BMP4, BMP6 and BMP7, influence esophageal squamous cancer cell (ESCC) growth, invasion and metastasis. BMP6 and type I receptor ALK2 and type II receptor BMPRII, ActRIIA and ActRIIB were expressed in all ESCC cell lines. In addition, adenovirus-mediated BMP overexpression did not affect ECA-109 cell growth. BMP6/7 overexpression increased ECA-109 cell invasion and metastasis, activated SMAD1/5/8 signal pathway and induced downstream gene ID1 expression. While BMP2/4 overexpression reduced ECA-109 cell invasion and metastasis and obviously promoted ERK1/2, P-38 and JNK activation with weak SMAD1/5/8 phosphorylation. When BMP6/7 favorite type I receptor ALK2 or type II receptor BMPRII was interfered with by dominant-negative mutation, BMP6/7-induced invasion and metastasis augmentation disappeared. Further investigation on clinical ESCC samples and non-tumorous adjacent tissue found that tumors with triple-positive BMP6, ALK2 and BMPRII had deeper growth than tumors with only BMP6 expression. These results suggested that different BMPs distinctly affected esophageal squamous cancer cell invasion and metastasis by employing different signal pathways.

BMP-6 inhibits the metastasis of MDA-MB-231 breast cancer cells by regulating MMP-1 expression.

Bone morphogenetic protein-6 (BMP-6) is a multifunctional molecule with distinct abilities in embryogenesis and organogenesis. In the present study, our results showed that the rate of BMP-6-negative expression was 30.56% in breast cancer tissues, but was 9.58% in normal tissues by immunohistochemical staining. This implied that BMP-6 expression is absent in breast cancer tissues and may suppress breast cancer metastasis. In addition, stable overexpression of BMP-6 in MDA-MB­231 cells was established to analyze the metastatic ability. The Boyden chamber assay showed that BMP-6 inhibited the migration and invasion of MDA-MB-231 cells. Moreover, real-time PCR analysis showed that BMP-6 markedly downregulated matrix metalloproteinase-1 (MMP-1) expression at both the mRNA and protein levels in the MDA-MB­231 cells. Importantly, the results of luciferase and CHIP assays revealed that BMP-6 inhibited MMP-1 promoter activity through the AP-1 response element. In MDA-MB-231 cells treated with BMP-6, a significant decrease in the recruitment of AP-1 components, c-Jun/c-Fos, to the endogenous MMP-1 promoter was noted. We also demonstrated that BMP-6 inhibited the invasion of MDA-MB-231 cells, and this effect was significantly attenuated by overexpression of MMP-1. In contrast, MMP-1 knockdown by RNA interference or MMP-1 inhibitor exhibited an opposite effect. These observations suggest a novel role of BMP-6 in the inhibition of breast cancer metastasis by regulating secretion of MMPs in the tumor microenvironment.

Effect of BMPs and Wnt3a co-expression on the osteogenetic capacity of osteoblasts.

In the present study, third­generation autologous­inactivated bone morphogenic protein 2 (BMP2), BMP4, BMP6, BMP7, BMP9 and Wnt3a lentiviral vectors were constructed and integrated into the genome of MC3T3­E1 murine mesenchymal stem cells (MMSCs) to produce osteoinductive factor gene­modified MMSCs. The transfection efficiency of each osteoinductive factor was then determined by detecting the expression levels of runt related transcription factor 2 (Runx2) mRNA. The cotransfection with combinations of two lentiviruses was performed, and the expression levels of bone γ­carboxyglutamate protein and alkaline phosphatase in the MC3T3­E1 cell culture supernatant were detected. The expression level of Runx2 mRNA was detected by reverse transcription­polymerase chain reaction, and western blotting was performed to detect the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a. The results demonstrated that the recombinant lentiviruses were successfully transfected into MC3T3­E1 cells. The relative expression levels of Runx2 mRNA were greatest in the BMP2 group, sequentially followed by the BMP4, BMP9, BMP7, Wnt3a and BMP6 groups. The results of cotransfection of MC3T3­E1 cells (a total of 8 groups) demonstrated that BMP­2 and BMP­7 exhibited the highest cotransfection efficiency. Western blot analysis demonstrated that following BMP2 and BMP7 cotransfection of MC3T3­E1 cells, the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a were increased compared with control cells. In conclusion, the third­generation lentiviral vectors effectively improved the osteogenic efficiencies of MC3T3­E1 cells, which provided an important theoretical basis and therapeutic strategy for bone reconstruction and tissue engineering.