Improved Soluble Expression in Escherichia coli and Easily Purified Recombinant Human Bone Morphogenetic 7-2 Fusion Protein.

BACKGROUND: Bone morphogenetic protein (BMP) is a cysteine-rich growth factor and plays a key role in early bone tissue development and bone defect repair. However, the low yield, high cost and complicated process in BMP significantly limit its clinical application. OBJECTIVE: In this study, we developed an efficient method for soluble expression and preparation of recombinant human bone morphogenetic 7-2 fusion protein (rhBMP7-2) and determined its molecular weight and biological activity. METHODS: The fusion gene for rhBMP-2 and rhBMP-7 was inserted into the pET-ELP expression vector. Correct DNA sequence was confirmed, the rhBMP7-2-ELP was transformed into Escherichia coli strain BL21 (DE3), and the rhBMP7-2 was produced in the recombinant E. coli. Recombinant BMP7-2 purify was identified using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The cell proliferation and biological activity of rhBMP7-2 were measured by Cell Counting Kit-8 and Alkaline Phosphatase assay using C2C12 cells, respectively. RESULTS: The result of digestion of NdeI, BamHI and XhoI enzymes showed that the rhBMP7-2-ELP was correctly constructed. The recombinant BMP7-2 was successfully expressed in soluble form; the purified rhBMP7-2 showed biological activity and significantly promoted cell proliferation and differentiation in a dose-dependent manner. CONCLUSION: The rhBMP7-2 fusion protein with osteogenic activity was prepared through a lowcost and time-efficient method. Our preparation method presents the potential to be applied to the large-scale production of rhBMP7-2 and is expected to play a significant role in clinical treatment.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Effects of BMP7 produced by group 2 innate lymphoid cells on adipogenesis.

Group 2 innate lymphoid cells (ILC2s) are type 2 cytokine-producing cells that have important roles in helminth infection and allergic inflammation. ILC2s are tissue-resident cells, and their phenotypes and roles are regulated by tissue-specific environmental factors. While the role of ILC2s in the lung, intestine and bone marrow has been elucidated in many studies, their role in adipose tissues is still unclear. Here, we report on the role of ILC2-derived bone morphogenetic protein 7 (BMP7) in adipocyte differentiation and lipid accumulation. Co-culture of fat-derived ILC2s with pluripotent mesenchymal C3H10T1/2 cells and committed white preadipocyte 3T3-L1 cells resulted in their differentiation to adipocytes and induced lipid accumulation. Co-culture experiments using BMP7-deficient ILC2s revealed that BMP7, produced by ILC2s, induces differentiation into brown adipocytes. Our results demonstrate that BMP7, produced by ILC2s, affects adipocyte differentiation, particularly in brown adipocytes.

Effects of Different Therapeutic Radiation Doses on the Development of Neural Tube Defects in Chick Embryos and the Correlation with Bone Morphogenetic Protein 4 and 7 Expression Levels.

AIM: To investigate the effects of different therapeutic radiation doses on the prevalence of neural tube defects (NTDs) in chick embryos and bone morphogenetic protein (BMP) 4 and BMP7 expression levels. MATERIAL AND METHODS: The chick embryos (n=143) were derived from fertile, specific pathogen-free eggs of domestic fowl. The presence of NTDs was analyzed using a stereomicroscope, and BMP4 and BMP7 expression levels were assessed by immunohistochemical staining. The chick embryos were divided into five groups: control (no radiation exposure) (n=23), exposure to thorax computerized tomography (CT) (n=30); exposure to abdominopelvic CT (n=30), exposure to cranium CT (n=30), and exposure to brain perfusion CT (n=30). RESULTS: The prevalence of NTDs and BMP4 and BMP7 expression levels in the different groups were compared. In the cranium CT dose group, both the NTD prevalence (20%, p=0.002) and BMP7 (p=0.031) expression levels were significantly higher than those in the other groups. However, none of the medical doses of irradiation altered BMP4 expression levels (p=0.242). No NTDs were detected in the thorax CT and abdominopelvic CT groups. CONCLUSION: Exposure to irradiation at cranium CT doses may induce the development of NTDs and increase BMP7 expression. Dose radiation exposure using thorax CT and abdominopelvic CT protocols does not appear to induce NTDs.

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Osteogenic protein-1 alleviates high glucose microenvironment-caused degenerative changes in nucleus pulposus cells.

Increasing evidence has indicated a close relationship between diabetes mellitus (DM) and disc degeneration. As a potential therapeutic growth factor, osteogenic protein-1 (OP-1) has lots of protective effects on the healthy disc cell's biology. The present study was aimed to investigate the effects of OP-1 on degenerative changes of nucleus pulposus (NP) cells in a high glucose culture. Rat NP cells were cultured in the baseline medium or the high glucose (0.2 M) culture medium. OP-1 was added into the high glucose culture medium to investigate whether its has some protective effects against degenerative changes of NP cells in the high glucose culture. NP cell apoptosis ratio, caspase-3/9 activity, expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3), matrix macromolecules (aggrecan and collagen II), and matrix remodeling enzymes (MMP-3, MMP-13, and ADAMTS-4), and immuno-staining of NP matrix proteins (aggrecan and collagen II) were evaluated. Compared with the baseline culture, high glucose culture significantly increased NP cell apoptosis ratio, caspase-3/9 activity, up-regulated expression of Bax, caspase-3, MMP-3, MMP-13 and ADAMTS-4, down-regulated expression of Bcl-2, aggrecan and collagen II, and decreased staining intensity of aggrecan and collagen II. However, the results of these parameters were partly reversed by the addition of OP-1 in the high glucose culture. OP-1 can alleviate high glucose microenvironment-induced degenerative changes of NP cells. The present study provides that OP-1 may be promising in retarding disc degeneration in DM patients.

BMP-7/Smad expression in dedifferentiated Schwann cells during axonal regeneration and upregulation of endogenous BMP-7 following administration of PTH (1-34).

PURPOSE:: To determine the expression and distribution of bone morphogenetic protein (BMP)-7 and related molecules during peripheral nerve regeneration and to assess whether administration of parathyroid hormone (PTH) drug (1-34) potentiates the intrinsic upregulation of BMP-7/Smad signaling. METHODS:: The rat sciatic nerves were crushed with an aneurysm clip resulting in axonal degeneration. In the normal nerve, and at 1, 2, 4, and 8 weeks after injury, BMP-7, BMP receptors, p-Smad 1/5/8, and Noggin, the endogenous BMP antagonist, were evaluated. Additionally, the distribution of BMP-7 was assessed by fluorescent double immunostaining. In vitro studies were also performed to examine the effect of BMP-7 and PTH (1-34) administration on rat Schwann cells (SCs). RESULTS:: Aneurysm clip made reliable animal model of the nerve injury with recovery at 8 weeks after the injury. BMP-7/Smad protein and mRNA were significantly upregulated on axon-SCs units at 1 week after injury, and this upregulated expression was maintained for 4 weeks. Besides, significant upregulation of Noggin's expression was observed on axon-SCs units at 2 weeks after injury. Moreover, fluorescent double immunostaining showed co-localization between expression of BMP-7 and p75NTR during axonal regeneration. In the in vitro study, administration of BMP-7 induced significant proliferation of SCs. Application of PTH (1-34) upregulated BMP-7 on SCs. DISCUSSION/CONCLUSION:: BMPs were reported to be involved in protection and recovery after injury as well as in neurogenesis. Our current study showed that BMP/Smad signaling molecules were upregulated on dedifferentiated SCs after peripheral nerve injury and that administration of BMP-7 increased SC viability in vitro. These results suggested that axonal regeneration could be induced via upregulation of endogenous BMP-7 on SCs by PTH (1-34) administration.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells.

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

BMP7 regulates lung fibroblast proliferation in newborn rats with bronchopulmonary dysplasia.

The present study investigated the expression of bone morphogenetic protein (BMP) 7 in a newborn rat model of bronchopulmonary dysplasia (BPD) and the biological effects of BMP7 on newborn rat lung fibroblast (LF) cells. For this purpose, a total of 196 newborn rats were randomly and equally assigned to a model group and a control group. Lung tissue was collected at days 3, 7, 14 and 21 for histological analysis. The location and expression of BMP7 was examined by immunohistochemical staining and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis. A total of 38 full­term newborn rats on the day of birth were sacrificed and LF cells were isolated and treated with BMP7. The biological effects of BMP7 on LF cells were assessed by cell proliferation and cell cycle analysis. The findings demonstrated that abnormal alveolar development due to BPD was gradually intensified in the model group over time. Immunohistochemical staining revealed that the location of BMP7 in lung tissue was altered. Immunohistochemistry and RT­qPCR assays demonstrated a gradual decrease in BMP7 expression in the model group induced by hyperoxia. MTT assays demonstrated that BMP7 inhibited LF cells and the inhibitory effect was dose­dependent and time­dependent. Flow cytometry revealed that the inhibitory effect of BMP7 in LF cells was causing cell cycle arrest at the G1 phase. The present study demonstrated that BMP7 may serve an important role in alveolar development in a BPD model. BMP7 may be involved in abnormal alveolar development through the regulation of LF proliferation.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-ß1 (TGF-ß1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (P < 0.05) and an improvement in the vascular disorganization rate (P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-ß1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization.

Protection of tubular epithelial cells during renal injury via post-transcriptional control of BMP7.

Severe injury of renal tubular epithelial cells may cause acute renal failure, the progression of which results in renal fibrosis, and obstructive nephropathy. Transforming growth factor ß 1 and bone morphogenic protein 7 (BMP7) play contradicting roles in and coordinate the process of epithelial-to-mesenchymal transition of renal tubular epithelial cells, but the molecular regulation of BMP7 remains ill-defined. Here, we addressed this question. We found that after induction of unilateral ureteral obstruction (UUO) in mice, the increases in BMP7 mRNA were much more pronounced than BMP7 protein in kidney, suggesting the presence of post-transcriptional control of BMP7. Moreover, significant increases in a BMP7-targeting microRNA, miR-384-5p, were detected in the mouse kidney post UUO. Overexpression of miR-384-5p significantly decreased BMP7 protein, while depletion of miR-384-5p significantly increased BMP7 protein in renal epithelial cells. Bioinformatics study showed that miR-384-5p appeared to suppress BMP7 protein translation, through its direct binding to the 3'-UTR of BMP7 mRNA. Furthermore, suppression of miR-384-5p in vivo attenuated severity of renal injury by UUO. Together, our study sheds light on miR-384-5p as a crucial factor that regulates the fibrosis-related pathogenesis after renal injury, and points to miR-384-5p as a promising innovative therapeutic target for prevention of renal fibrosis.

Acceleration of bone regeneration in bioactive glass/gelatin composite scaffolds seeded with bone marrow-derived mesenchymal stem cells over-expressing bone morphogenetic protein-7.

In this research, the osteoinduction effect of a novel variant of bone morphogenetic protein-7 (BMP-7), delivered through bone marrow mesenchymal stem cells (BM-MSCs) seeded on bioactive glass/gelatin nanocomposite scaffolds, was evaluated in a calvarial critical size defect in rats. After being harvested and characterized in vitro, BM-MSCs were infected by a plasmid vector containing BMP-7 encoding gene enriched with a heparin-binding site (B2BMP-7) to assess its osteogenic effects in vivo. The animals were randomly categorized into three groups receiving the scaffold alone (group I), the scaffold seeded with BM-MSCs (group II), and the scaffold seeded with manipulated BM-MSCs (group III). After 2, 4 and 12 postoperative weeks, the animals were sacrificed and the harvested specimens were analyzed using histological and immunohistochemical staining. The results of in vitro tests (preliminary screening) showed that the synthesized scaffolds were biocompatible constructs supporting cell attachment and expansion. The in vivo results revealed higher osteogenesis in the defects filled with the B2BMP-7 excreting BM-MSCs/scaffolds compared to the other two groups. After 12weeks of implantation, fully mature newly formed bone was detected throughout the damaged site, which indicates a synergistic effect of cells, scaffolds and growth factors in the process of tissue regeneration. Therefore, bioactive glass-containing scaffolds pre-seeded with manipulated BM-MSCs exhibit an effective combination to improve osteogenesis in bone defects, and the approach followed in this work could have a significant impact in the development of novel tissue engineering constructs.

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.

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.

hBMP-7 induces the differentiation of adipose-derived mesenchymal stem cells into osteoblast-like cells.

The aim of this study was to investigate the differentiation potential of adipose-derived mesenchymal stem cells (ADMSCs) into osteoblasts by human bone morphogenetic protein-7 (hBMP-7) induction. ADMSCs were isolated from the subcutaneous adipose tissue of a rabbit, and then transfected with the pcDNA3.1 vector alone and pcDNA3.1-hBMP-7 (hBMP-7), respectively. Untransfected ADMSCs were used as the control group. After transfection, the morphology and green fluorescent protein (GFP) fluorescence intensity of ADMSCs were observed by fluorescent microscopy. The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the growth of ADMSCs at 1, 3, and 5 days, respectively. Transmission electron microscopy was performed to observe the ultrastructural morphology of ADMSCs. In addition, ADMSCs were stained with quinalizarin and toluidine blue to reflect the content of osteoblasts and chondrocytes, respectively. Finally, the expression of collagen I and osteocalcin in ADMSCs was detected by western blot. ADMSCs were successfully isolated. Obvious GFP fluorescence and high expression of hBMP-7 demonstrated the successful transfection of hBMP-7. Specific morphological characters with a metabolically active ultrastructure were exhibited on the ADMSCs transfected with hBMP- 7. In addition, the growth rate of ADMSCs transfected with hBMP-7 was significantly higher than that of the cells in the vector and control groups. Successfully induced osteoblast-like cells were identified by an obvious erythrine area and high expression of collagen I and osteocalcin in ADMSCs transfected with hBMP-7. Thus, ADMSCs can be successfully differentiated into osteoblast-like cells by hBMP-7 induction in vitro.

Simvastatin Treatment Upregulates Anti-Fibrotic Bone Morphogenetic Protein-7 Expression at Rat Cardiac Allograft Rejection.

BACKGROUND: Bone morphogenetic protein (BMP)-7 mediates ischemic tolerance and anti-fibrotic effects in various organs such as kidney and heart. Recently, reno- and podocyte-protective effects of a potent HMG-CoA reductase inhibitor, pitavastatin, were accompanied by BMP-7 upregulation. METHODS: Here, we investigated the effect of simvastatin treatment on BMP-7 expression in major MHC-mismatched rat cardiac allografts subjected to ischemia-reperfusion injury and adaptive immune activation at 10 days. RESULTS: We localized Smad2 activity and Reca-1+ fibroblast specific protein-1+ immunoreactivity, suggesting endothelial-to-mesenchymal transition, at fibrotic borderline of cardiac allografts at 10 days. Simvastatin donor and recipient combination treatment significantly upregulated cardiac allograft BMP-7 expression when compared to nontreated controls at 10 days. CONCLUSION: The beneficial effect of statin treatment on cardiac allograft may in part be mediated through the upregulation of BMP-7.

Attenuation of liver fibrosis by herbal compound 861 via upregulation of BMP-7/Smad signaling in the bile duct ligation model rat.

Herbal compound 861 (Cpd 861) exerts an anti-fibrotic effect in patients with hepatic fibrosis; however, the anti-fibrotic mechanism has yet to be fully elucidated. The present study aimed to explore the mechanistic basis for the anti-fibrotic effect, with a focus on bone morphogenetic protein 7 (BMP-7)/Smad signaling in a bile duct ligation (BDL)-induced liver fibrosis rat model. Following the induction of hepatic fibrosis, rats induced by BDL were treated with 9 g/kg Cpd 861 daily or an equal volume of saline for 28 days. Serum samples were prepared for monitoring the levels of alanine transaminase, aspartate transaminase and total bilirubin, and direct bilirubin analyses and liver samples were used to investigate gene expression, protein localization and protein expression analysis using real­time quantitative polymerase chain reaction, immunohistochemistry and western blotting. The results revealed the attenuation of liver fibrosis by Cpd 861 in the histological and biochemical experiments. BMP­7 and phospho (p)­Smad1/5/8 were localized predominantly in the cytoplasm of hepatocytes. In comparison with the saline­treated BDL rats, Cpd 861 markedly upregulated the gene expression of BMP­7 and Smad5, as well as the protein expression of BMP­7 and Smad1/5. In addition, p-Smad1/5/8 protein expression was markedly increased by Cpd 861 in the BDL model. These results indicated that Cpd 861 alleviates hepatic fibrosis possibly via the upregulation and activation of BMP-7/Smad signaling in hepatic fibrotic rats.

Artesunate attenuates unilateral ureteral obstruction-induced renal fibrosis by regulating the expressions of bone morphogenetic protein-7 and uterine sensitization-associated gene-1 in rats.

PURPOSE: Kidney fibrosis is the most common final stage of progressive renal disease. Bone morphogenetic protein-7 (BMP-7) has been shown to be important in both preservation of kidney function and resistance to injury. Recently, it has been realized that uterine sensitization-associated gene-1 (USAG-1) functions as a kidney-specific BMP antagonist. Because of the reported anti-fibrotic effects of artesunate (Art), this study was designed to investigate the effects of Art on renal fibrosis in unilateral ureteral obstruction (UUO) rats and to explore the underlying mechanisms. METHODS: Thirty male Sprague-Dawley rats were randomly divided into five groups: Sham group, UUO group, low-dose Art-treated (Art-L), middle-dose Art-treated (Art-M), and high-dose Art-treated (Art-H) groups. The UUO rat model was established by ligating the left ureter. Fourteen days later, interstitial collagen deposition, expression of USAG-1, BMP-7, E-cadherin, α-smooth muscle actin (α-SMA), fibronectin (FN), collagen I, as well as the inflammatory infiltration levels in the kidneys were assessed. RESULTS: Art treatment significantly attenuated the deposition of interstitial collagens in the UUO rats' kidneys and exhibited the ability to improve renal function, followed by the up-regulated expression of BMP-7 and E-cadherin and the down-regulated expression of USAG-1 and α-SMA. In addition, increased macrophages infiltration in the kidneys of the UUO rats were also attenuated by the administration of Art. CONCLUSIONS: These results indicate that Art is able to improve the renal function decline and renal fibrosis induced by UUO, which may be associated with the up-regulation of BMP-7 and down-regulation of USAG-1. Accordingly, Art may become a potential preventive or therapeutic agent for chronic kidney diseases.

Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of tumor and is associated with high morbidity and mortality rates. Patients with HCC routinely undergo surgery followed by adjuvant radiation therapy and chemotherapy. Despite such aggressive treatment approaches, median survival times remain under 1 year in most cases. KDM5C is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). KDM5C has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in HCC remain unclear. METHODS: Expression level of KDM5C was examined by RT-PCR, and IHC. Forced expression of KDM5C was mediated by retroviruses, and KDM5C was downregulated by shRNAs expressing lentiviruses. Migration and invasion of HCC cells was measured by wound healing, Transwell and Matrigel assays respectively. RESULTS: In this study, we report that KDM5C is abundantly expressed in invasive human HCC cells. Cellular depletion of KDM5C by shRNA inhibited HCC cell migration, invasion and epithelial-mesenchymal transition in vitro, and markedly decreased the metastasis capacity of invasive HCC cells in the liver and lung. Furthermore, ectopic expression of KDM5C in HCC cells promoted cell migration, invasion and epithelial-mesenchymal transition via the inactivation of BMP7. Knockdown of BMP7 significantly promotes shKDM5C-induced cell migration inhibition. CONCLUSIONS: Taken together, these data suggest that KDM5C-mediated BMP7 inactivation is essential for HCC cell invasion.

Expression of recombinant BMP-7 gene increased ossification activity in the rabbit bone mesenchymal stem cells.

OBJECTIVE: The mesenchymal stem cells (MSCs), which were distributed in the bone marrow stroma, become ideal progenitor cells in bone tissue engineering because of their convenient isolation, small injury when obtained, and strong osteogenic capacity. The osteogenic differentiation of MSCs, which is indicated by the increased alkaline phosphatase (ALP) activity and the enhanced accumulation of collagen, could be induced by a strong osteogenic capacity biological factor termed bone morphogenetic protein-7 (BMP-7). Although the chemically synthesized BMP-7 was widely applied to study the osteogenic differentiation of MSCs, transferring and expressing BMP-7 gene in target cells is more desirable, especially for gene therapy, given the advantages and convenience on the stable expression of BMP-7. The aim of this study was to determine whether recombinant BMP-7-expressing MSCs would induce bone formation in vitro. MATERIALS AND METHODS: BMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying BMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. MSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-BMP-7-transfected, pcDNA3.1-transfected, and untransfected. Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University. RESULTS: A significant increase of ALP activity was detected in the supernatant of pcDNA3.1-BMP-7 transfected MSCs, and the enhanced collagen accumulation, which was inferred by the increased hydroxyproline content and RT-PCR. CONCLUSIONS: These results implied that BMP-7 gene was expressed in MSCs sufficiently and was involved in inducing differentiation of MSCs into osteoblast.