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.

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.

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.

Bone morphogenetic protein-inducer tilorone identified by high-throughput screening is antifibrotic in vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-ß signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-ß signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

Effect of NSAIDs and diuretics on nephrogenesis in an ex vivo embryogenic kidney model.

The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole-mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short-term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.

Osteogenesis induced by a bone forming peptide from the prodomain region of BMP-7.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Many researchers have tried to develop adjuvants as specific suppressors of bone resorption and stimulators of bone formation for therapeutic purposes in patients with osteoporosis. Therefore, specific stimulators on bone formation are one of therapeutic significance in the treatment of osteoporosis. Until now, the regulation of bone generation has been the focus of bone morphogenetic protein-7 (BMP-7) investigation from mature form. However, new peptides from immature form which has osteogenic activity has not been reported and developments of these proteins are still remained. In this study, we found a new peptide sequence, called bone forming peptide-1 (BFP-1) and have more high activities of osteogenic differentiation compared with BMP-7. BFP-1-treated multipotent bone marrow stromal stem cells (MBSCs) induced the expression levels and activity of alkaline phosphatase (ALP). Moreover, BFP-1 enhanced the levels of CD44, CD47 and CD51 expression as well as increased Ca(2+) content in MBSCs. In current study, radiography at 8 weeks revealed that BFP-1 pretreated-MBSC transplanted animals had strongly increased bone formation compared to that in the BMP-7 pretreated MBSC transplanted animals. Our finding indicates a new insight into peptides from the immature region of BMP-7 can also be useful in the development of adjuvant therapies for bone-related diseases.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium.

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.