The Regulation of Collagen Processing by miRNAs in Disease and Possible Implications for Bone Turnover

This article describes several recent examples of miRNA governing the regulation of the gene expression involved in bone matrix construction. We present the impact of miRNA on the subsequent steps in the formation of collagen type I. Collagen type I is a main factor of mechanical bone stiffness because it constitutes 90–95% of the organic components of the bone. Therefore, the precise epigenetic regulation of collagen formation may have a significant influence on bone structure. We also describe miRNA involvement in the expression of genes, the protein products of which participate in collagen maturation in various tissues and cancer cells. We show how non-collagenous proteins in the extracellular matrix are epigenetically regulated by miRNA in bone and other tissues. We also delineate collagen mineralisation in bones by factors that depend on miRNA molecules. This review reveals the tissue variability of miRNA regulation at different levels of collagen maturation and mineralisation. The functionality of collagen mRNA regulation by miRNA, as proven in other tissues, has not yet been shown in osteoblasts. Several collagen-regulating miRNAs are co-expressed with collagen in bone. We suggest that collagen mRNA regulation by miRNA could also be potentially important in bone metabolism.

Methylpiperidinopyrazole Attenuates Estrogen-Induced Mitochondrial Energy Production and Subsequent Osteoblast Maturation via an Estrogen Receptor Alpha-Dependent Mechanism

An estrogen deficiency is the main cause of osteoporosis in postmenopausal women. In bone remodeling, estrogen receptors (ERs) can mediate estrogen-transducing signals. Methylpiperidinopyrazole (MPP) is a highly specific antagonist of ER-alpha (ERα). This study was designed to evaluate the effects of MPP on estrogen-induced energy production, subsequent osteoblast maturation, and the possible mechanisms. Exposure of primary osteoblasts isolated from neonatal rat calvarias to MPP did not affect cell morphology or survival. Estradiol can induce translocation of ERα into mitochondria from the cytoplasm. Interestingly, pretreatment of rat calvarial osteoblasts with MPP lowered estrogen-induced ERα translocation. Sequentially, estrogen-triggered expressions of mitochondrial energy production-linked cytochrome c oxidase (COX) I and COX II messenger (m)RNAs were inhibited following pretreatment with MPP. Consequently, MPP caused decreases in estrogen-triggered augmentation of the activities of mitochondrial respiratory complex enzymes and levels of cellular adenosine phosphate (ATP). During progression of osteoblast maturation, estrogen induced bone morphogenetic protein (BMP)-6 and type I collagen mRNA expressions, but MPP treatment inhibited such induction. Consequently, estrogen-induced osteoblast activation and mineralization were attenuated after exposure to MPP. Taken together, MPP suppressed estrogen-induced osteoblast maturation through decreasing chromosomal osteogenesis-related BMP-6 and type I collagen mRNA expressions and mitochondrial ATP synthesis due to inhibiting energy production-linked COX I and II mRNA expressions. MPP can appropriately be applied to evaluate estrogen-involved bioenergetics and osteoblast maturation.

P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.

Plumbagin Alleviates Capillarization of Hepatic Sinusoids In Vitro by Downregulating ET-1, VEGF, LN, and Type IV Collagen

Critical roles for liver sinusoidal endothelial cells (LSECs) in liver fibrosis have been demonstrated, while little is known regarding the underlying molecular mechanisms of drugs delivered to the LSECs. Our previous study revealed that plumbagin plays an antifibrotic role in liver fibrosis. In this study, we investigated whether plumbagin alleviates capillarization of hepatic sinusoids by downregulating endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), laminin (LN), and type IV collagen on leptin-stimulated LSECs. We found that normal LSECs had mostly open fenestrae and no organized basement membrane. Leptin-stimulated LSECs showed the formation of a continuous basement membrane with few open fenestrae, which were the features of capillarization. Expression of ET-1, VEGF, LN, and type IV collagen was enhanced in leptin-stimulated LSECs. Plumbagin was used to treat leptin-stimulated LSECs. The sizes and numbers of open fenestrae were markedly decreased, and no basement membrane production was found after plumbagin administration. Plumbagin decreased the levels of ET-1, VEGF, LN, and type IV collagen in leptin-stimulated LSECs. Plumbagin promoted downregulation of ET-1, VEGF, LN, and type IV collagen mRNA. Altogether, our data reveal that plumbagin reverses capillarization of hepatic sinusoids by downregulation of ET-1, VEGF, LN, and type IV collagen.