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Previous studies have demonstrated that transcriptional silencing of miRNAs due to DNA hypermethylation is associated with different pathologies. It has also been reported that abnormal expression of miR-140-5p and miR-146a is linked to osteoarthritis (OA) progression. In this study, we investigated the role of DNA methylation on miR-140-5p and miR-146a expression in OA. miR-140-5p and miR-146a expression was investigated by qRT-PCR. The methylation status of miR-140 and miR-146a regulatory regions was analyzed using qMSP and bisulfite sequencing analysis. SMAD-3 and NF-kB binding to miR-140 and miR-146a regulatory regions was assessed by ChIP assay and knockdown experiments. OA-related genes' expression was evaluated in 5-AzadC, miRNAs inhibitor and 5-AzadC/miRNAs inhibitor-treated cells. Hypermethylation of specific CpG sites in miR-140 and miR-146a regulatory regions was associated with downregulation of miR-140-5p and miR-146a in OA chondrocytes and synoviocytes, respectively. 5-AzadC-induced miR-140-5p and miR-146a upregulation was observed in OA chondrocytes and synoviocytes. Moreover, we found decreased binding affinity of SMAD-3 and NF-kB transcription factors on the hypermethylated miR-140-5p and miR-146a regulatory regions, respectively. Downregulation of MMP-13 and ADAMTS-5 in 5-AzadC-treated OA chondrocytes was prevented by miR-140-5p inhibitor transfection. Similarly, 5-AzadC-treated OA synoviocytes showed decreased expression of IRAK-1, IL1Β and IL-6, which was reversed following 5-AzadC-/miR-146a inhibitor treatment. Our results strongly suggest the impact of DNA methylation on miR-140-5p and miR-146a suppression in OA chondrocytes and synoviocytes, contributing to OA pathogenesis. [ABSTRACT FROM AUTHOR]

Background. Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. Methods. Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). Results. We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. Discussion. On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell's basic antioxidant machinery. [ABSTRACT FROM AUTHOR]

Abstract: p53 regulates several biological processes, including senescence. Its protein stability is regulated by ubiquitination and proteasomal degradation, mainly mediated by Mdm2. However, other E3 ligases have been identified, such as the chaperone-associated ligase CHIP, although their precise function regarding p53 degradation remains elusive. Interestingly, CHIP deficiency has been recently shown to result in accelerated aging in mice, although the molecular basis of this phenotype was not completely understood. In this study, we explore the role of CHIP in regulating p53 in senescence. We demonstrate that in senescent human fibroblasts, CHIP is up-regulated concomitant with a significant down-regulation of p53. Moreover, CHIP partially translocates to the nucleus and acquires higher ubiquitination levels in senescent cells. Notably, CHIP overexpression in young cells, to levels similar to those recorded during senescence, leads to p53 degradation to below its basal levels. In addition, whereas CHIP silencing has no effect on p53 stability in young cells, a considerable p53 accumulation occurs in their senescent counterparts. Finally, we have observed an attenuation of the CHIP-associated molecular folding–refolding machinery during senescence, and supportively, inhibition of Hsp90 activity leads to rapid p53 degradation only in senescent cells. Taking these results together, we conclude that CHIP-dependent p53 regulation occurs specifically during senescence. [ABSTRACT FROM AUTHOR]

Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties.In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([γ-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS–PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls.These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes. [Copyright &y& Elsevier]

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non-phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied. [ABSTRACT FROM AUTHOR]

It has been reported that oxidative stress (OS) is involved in the pathogenesis of osteoarthritis (OA) and that defective autophagy/mitophagy is accompanying the OA phenotype. In this study, we analyzed the effect of acute exogenous oxidative insult in control and OA chondrocytes. We analyzed the mRNA and protein levels of genes (Atg5, Beclin-1 and LC3) known to be involved in the formation of the autophagosome in control and OA cells treated with sublethal concentrations of hydrogen peroxide (H2O2). Cells were allowed to recover and the same analyses were performed. Under these conditions morphological differences in the mitochondria of control and OA cells were also assessed. Our results showed that the expression of these genes was significantly reduced in OA cells under normal conditions. Furthermore, in control cells mRNA and protein levels of these genes increased as a result of the OS and decreased back to their original levels 24 h after the treatment. On the contrary, their levels remained high in OA cells even after 24 h. Also, a significantly higher number of impaired mitochondria were observed in OA cells as compared to control cells at all time points. These data offer further understanding on the role of autophagy in response to OS in the onset and progression of OA. [ABSTRACT FROM AUTHOR]

Abstract It has been reported that oxidative stress (OS) is involved in the pathogenesis of osteoarthritis (OA) and that defective autophagy is accompanying this age-related disease. Moreover, it has been proposed that induction of autophagy could serve as therapeutic mean, as it was shown to alleviate several symptoms in OA animal models. On the contrary, it is also known that autophagic death, which results from over-activation of autophagy, is also a contributor in the development of this disease. Given this discrepancy, in this study we aimed at analysing the autophagic response against acute exogenous oxidative insult of chondrocytes from healthy individuals (control) and OA patients (OA). Cells were treated with sublethal concentrations of hydrogen peroxide (H 2 O 2) and then allowed to recover for different periods of time. Firstly, mRNA levels of autophagy-related genes (ATG5, Beclin-1 and LC3) were found significantly reduced in OA chondrocytes compared to control chondrocytes under physiological conditions. After the exposure to OS, in control cells mRNA and protein levels of these genes initially increased and decreased back to their basal levels 6–24 h after treatment. On the contrary, in OA chondrocytes the levels of autophagy-related genes remained high even 24 h post-treatment, indicating their inability to attenuate autophagy. Under the same conditions, the staining pattern of LC3, known marker of autophagosome formation, was analysed, and possible morphological differences between mitochondria of control and OA cells were microscopically assessed. These analyses revealed higher number of impaired mitochondria as well as increased autophagosome formation in OA cells as compared to control cells at all time points. Taken together, our results demonstrate a deregulation of the autophagic response against the oxidative insult in OA chondrocytes and offers insights on autophagy's role in the progression of OA. Graphical abstract fx1 Highlights • This paper highlights the involvement of autophagy in osteoarthritis (OA). • The oxidative pressure onto OA chondrocytes leads to autophagy reduction and damage. • It also leads to mitochondria malformation that drives autophagosomes formation. • This creates a pre-autophagic pool in OA chondrocytes that deregulates autophagy. • These results are challenging the concept of autophagy induction in OA therapy. [ABSTRACT FROM AUTHOR]