Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.     

Factors influencing myofibroblast differentiation during wound healing and fibrosis.

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilaments bundles and the expression of α-SM actin, the actin isoform typical of contractile vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When granulation tissue evolves into a scar, myofibroblasts containing α-SM actin disappear, probably as a result of apoptosis. In contrast, myofibroblasts expressing α-SM actin persist in excessive scarring and in fibrotic conditions. The mechanisms leading to the development of myofibroblastic features remain to be investigated. Studies on the factors regulating the phenotype of myofibroblasts will be necessary for understanding their behavior in vivo, and possibly modifying this behavior during the different clinical settings.     

MiR-7b directly targets DC-STAMP causing suppression of NFATc1 and c-Fos signaling during osteoclast fusion and differentiation

DC-STAMP is a key regulating molecule of osteoclastogenesis and osteoclast precursor (OCP) fusion. Emerging lines of evidence showed that microRNAs play crucial roles in bone metabolism and osteoclast differentiation, but no microRNA has yet been reported to be directly related to OCPs fusion. Through a microarray, we found that the expression of miR-7b in RAW264.7 cells was significantly decreased after induction with M-CSF and RANKL. The overexpression of miR-7b in RAW264.7 cells attenuated the number of TRAP-positive cells number and the formation of multinucleated cells, whereas the inhibition of miR-7b enhanced osteoclastogenesis. Through a dual luciferase reporter assay, we confirmed that miR-7b directly targets DC-STAMP. Other fusogenic molecules, such as CD47, ATP6v0d2, and OC-STAMP, were detected to be down-regulated in accordance with the inhibition of DC-STAMP. Because DC-STAMP also participates in osteoclast differentiation through the ITAM-ITIM network, multiple osteoclast-specific genes in the ITAM-ITIM network were detected to identify how DC-STAMP is involved in this process. The results showed that molecules associated with the ITAM-ITIM network, such as NFATc1 and OSCAR, which are crucial in osteoclastogenesis, were consistently altered due to DC-STAMP inhibition. These findings suggest that miR-7b inhibits osteoclastogenesis and cell-cell fusion by directly targeting DC-STAMP. In addition, the inhibition of DC-STAMP and its downstream signals changed the expression of other fusogenic genes and key regulating genes, such as Nfatc1, c-fos, Akt, Irf8, Mapk1, and Traf6. In conclusion, our findings indicate that miR-7b may be a potential therapeutic target for the treatment of osteoclast-related bone disorders.     

Gene expression of flap endonuclease-1 during cell proliferation and differentiation

It has been shown that flap endonuclease-1 (FEN-1), a structure-specific nuclease, acts on the removal of RNA primers during Okazaki fragment maturation in DNA synthesis. To study whether the gene expression of FEN-1 is inducible during cell proliferation, we analyzed the FEN-1 mRNA levels in actively growing cells and non-growing cells. The gene expression of FEN-1 was higher in mitotic cells than in resting cells, and was markedly decreased, especially, when terminal differentiation was induced in promyelocytic leukemia cells (HL-60 cells). The decline correlated substantially with the ceasing of DNA synthesis. In the examination of tissue-specific gene expression, the human testis, spleen, thymus and mucosal lining of colon tissues expressed this gene actively, whereas the prostate, ovary, small intestine and peripheral blood leukocyte hardly expressed it. In addition, FEN-1 was co-localized with the proliferating cell nuclear antigen (PCNA) in young rat kidney according to immunohistochemistry. These findings suggest that FEN-1 gene expression is inducible during cell proliferation for DNA synthesis, and is down-regulated during cell differentiation.     

The paracaspase MALT1 controls caspase-8 activation during lymphocyte proliferation

Caspase-8, an initiator caspase involved in lymphocyte apoptosis, is paradoxically required for lymphocyte proliferation. It is not understood how caspase-8 is controlled during antigenic signaling to allow for activation while averting the triggering of apoptosis. Here, we show that caspase-8 undergoes limited activation upon antigenic stimulation, and this activation is dependent on the paracaspase MALT1. The paracaspase domain of MALT1, in a protease-independent manner, induces caspase-8 activation through direct association. MALT1 diminishes the activation of apoptotic effector caspases, but it does not alter the activity of caspase-8 toward c-FLIP(L), which is required for antigenic signaling. Mutants of MALT1 that fail to activate caspase-8 and permit c-FLIP(L) cleavage cannot facilitate NF-kappaB activation or IL-2 induction. Our results reveal a mechanism that utilizes a protease potentially deadly to the cell for proliferative signaling and demonstrate a functional connection between the caspase and paracaspase families to enable nonapoptotic processes.     

The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein

Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Role of endothelin receptors on basal and endothelin-1-stimulated lung myofibroblast proliferation

Proliferation of myofibroblasts (MYF) contributes to numerous lung disorders. Endothelin-1 (ET-1) production is increased in various lung diseases and could contribute to lung remodelling. The respective roles of ETA and ETB receptors (ETA-R, ETB-R) and the role of endogenous ET-1 production by lung MYF on proliferation of MYF remain uncertain. Rat lung MYF were isolated and 3H-thymidine and 3H-leucine incorporation assays were completed in the presence of a selective ETA-R antagonist, a selective ETB-R antagonist, or a combination of both. Receptor expression was evaluated by confocal imaging, and ET-1 levels were measured by ELISA. Confocal microscopy revealed abundant ETA-R and ETB-R expression on lung MYF. ET-1 (10 nmol/L) stimulated MYF proliferation and protein synthesis through PI3-kinase and p38 pathways. Although neither selective ETA-R blockade (BQ-123, 1 micromol/L) nor selective ETB-R blockade (BQ-788, 1 micromol/L) alone inhibited proliferation or protein synthesis, their combination almost completely abolished ET-1 mitogenic effect. Surprisingly, basal MYF proliferation was increased by selective blockade of either ETA-R or ETB-R alone, but not by dual blockade. ET-1 levels were not affected by the antagonists. Our findings indicate that both the ETA-R and the ETB-R regulate basal and stimulated lung MYF proliferation and suggest possible interactions between the receptors.     

Differential and combined effects of cardiotrophin-1 and TGF-beta1 on cardiac myofibroblast proliferation and contraction

Myofibroblasts respond to an array of signals from mitogens and cytokines during the course of wound healing following a myocardial infarction (MI), and these signals may coordinate ventricular myofibroblast proliferation. Furthermore, myofibroblasts are contractile and contribute to wound contraction by imparting mechanical tension on surrounding extracellular matrix. Although TGF-beta(1), CT-1, and PDGF-BB participate in various stages of post-MI wound healing, their combined net effect(s) on myofibroblast function is unknown. We investigated myofibroblast proliferation, expression of cell cycle proteins, and contractile function of cells treated with TGF-beta(1) and/or CT-1. We confirmed that TGF-beta(1) (10 ng/ml) suppresses proliferation of these cells, whereas CT-1 (10 ng/ml) and, for comparative purposes, PDGF-BB (1 ng/ml) treatments were associated with proliferation. Specific TGF-beta(1) treatment ablated CT-1-induced myofibroblast proliferation. TGF-beta(1) effects were specific, as they were suppressed by either TGF-beta-neutralizing antibody or viral Smad7 overexpression. TGF-beta(1) treatment also increased expression of p27 and decreased expression of cyclin E and Cdk2 in primary cells. CT-1 (10 ng/ml) treatment of myofibroblasts had no effect on collagen gel deformation versus controls, whereas TGF-beta(1) (10 ng/ml) and PDGF (10 ng/ml) treatments were associated with significant cell contraction; again, TGF-beta(1)-mediated contraction was unaffected by CT-1. Alone, CT-1 and TGF-beta(1) treatments exert opposing effects on myofibroblast function, whereas in combination TGF-beta(1)-mediated effects supersede those of CT-1 (and PDGF-BB). Thus TGF-beta(1) and CT-1 exert differential effects on myofibroblast proliferation and contraction in vitro, and we suggest that a balance of these effects may be important for the execution of normal cardiac wound healing.     

Degradation of IF1 controls energy metabolism during osteogenic differentiation of stem cells

Differentiation of human mesenchymal stem cells (hMSCs) requires the rewiring of energy metabolism. Herein, we demonstrate that the ATPase inhibitory factor 1 (IF1) is expressed in hMSCs and in prostate and colon stem cells but is not expressed in the differentiated cells. IF1 inhibits oxidative phosphorylation and regulates the activity of aerobic glycolysis in hMSCs. Silencing of IF1 in hMSCs mimics the metabolic changes observed in osteocytes and accelerates cellular differentiation. Activation of IF1 degradation acts as the switch that regulates energy metabolism during differentiation. We conclude that IF1 is a stemness marker important for maintaining the quiescence state.     

Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.     

miR-542-3p suppresses osteoblast cell proliferation and differentiation,targets BMP-7 signaling and inhibits bone formation

MicroRNAs (miRNAs) are short non-coding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes. Recent studies have suggested that miRNAs might have a role in osteoblast differentiation and bone formation. Here, we show that miR-542-3p, a well-characterized tumor suppressor whose downregulation is tightly associated with tumor progression via C-src-related oncogenic pathways, inhibits osteoblast proliferation and differentiation. miRNA array profiling in Medicarpin (a pterocarpan with proven bone-forming effects) induced mice calvarial osteoblast cells and further validation by quantitative real-time PCR revealed that miR-542-3p was downregulated during osteoblast differentiation. Over-expression of miR-542-3p inhibited osteoblast differentiation, whereas inhibition of miR-542-3p function by anti-miR-542-3p promoted expression of osteoblast-specific genes, alkaline phosphatase activity and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3′ UTR reporter assay identified BMP-7 (bone morphogenetic protein 7) as a direct target of miR-542-3p. It was seen that over-expression of miR-542-3p leads to repression of BMP-7 and inhibition of BMP-7/PI3K- survivin signaling. This strongly suggests that miR-542-3p suppresses osteogenic differentiation and promotes osteoblast apoptosis by repressing BMP-7 and its downstream signaling. Furthermore, silencing of miR-542-3p led to increased bone formation, bone strength and improved trabecular microarchitecture in sham and ovariectomized (Ovx) mice. Although miR-542-3p is known to be a tumor repressor, we have identified second complementary function of miR-542-3p where it inhibits BMP-7-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-542-3p by anti-miR-542-3p could represent a therapeutic strategy for enhancing bone formation in vivo.     

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.