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Downregulation of angiopoietin-like protein 2 inhibits cementoblast differentiation partially by activating the ERK1/2 signaling pathway.

ABSTRACT: Angiopoietin-like protein 2 (ANGPTL2) is abundantly expressed in adipose tissue, is associated with tissue homeostasis, and promotes osteoblast and chondrocyte differentiation. In teeth, cementum, a thin layer of mineralized tissue that is formed by cementoblasts, covers the entire root surface and is a vital component of periodontium. The cementoblasts regulate the deposition and mineralization of the cementum matrix. However, the effects of ANGPTL2 on cementoblast differentiation have not been studied. The objective of this study was to elucidate the role of ANGPTL2 during cementoblast differentiation and determine its underlying mechanisms. Our results showed that the expression levels of ANGPTL2 gradually increased during cementoblast differentiation. After ANGPTL2 was knocked down using short-hairpin RNA, the levels of the osteogenic markers osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) decreased. In addition, ALP activity and the number of calcified nodules were dramatically reduced compared with those in the negative control. Interestingly, the ERK1/2 signaling pathway was activated after ANGPTL2 knockdown. Treatment with PD98059, the inhibitor of the ERK1/2 signaling pathway, partially rescued the decreased differentiation capability of cementoblast caused by ANGPTL2 downregulation. Collectively, ANGPTL2 knockdown inhibited cementoblast differentiation partially by activating the ERK1/2 signaling pathway. These findings suggest that ANGPTL2 was indispensable in cementoblast differentiation.

SUBMITTER: Jiang C

PROVIDER: S-EPMC6357320 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Activated macrophages serve a key role in various inflammatory diseases, such as atherosclerosis and septic shock. Tripartite motif-containing protein 65 (TRIM65) has been previously reported to participate in tumor progression and lung inflammation. However, the molecular mechanisms that controls its expression under inflammatory conditions and its consequences in activated macrophages remain poorly understood. The present study first collected the tissues of C57BL/6J mice, smooth muscle cells, macrophages and endothelial cells to determine the expression and distribution of TRIM65 by reverse transcription-quantitative (RT-q) PCR and western blotting. Mouse and human macrophages were treated with LPS and C57BL/6J mice were intraperitoneally injected with LPS followed by isolation of spleen, lung, aorta and bone marrow. Following treatment, TRIM65 mRNA and protein level was examined by RT-qPCR and western blotting. The results showed that TRIM65 was highly expressed in organs of the immune system, such as the spleen, lymph node and thymus, but lowly expressed in heart, liver, brain and kidneys. TRIM65 was also highly expressed in macrophages and endothelial cells. TRIM65 mRNA and protein expression levels were found to be decreased in LPS-treated macrophages in vitro and in tissues isolated from C57BL/6J mice intraperitoneally injected with LPS in vivo. In addition, to identify the signaling pathways by which LPS regulates TRIM65 expression, inhibitors of MAPK and Akt signaling pathways were used to treat macrophages followed by examination the expression of TRIM65 by western blotting. The results demonstrated that LPS-inhibited TRIM65 expression was blocked by treatment with the ERK1/2 inhibitor U0126. Moreover, the RT-qPCR results showed that TRIM65 knockout potentiated LPS-induced expression of inflammatory cytokines in macrophages. Taken together, data from the present study suggest that LPS decreased TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway, whilst TRIM65 knockout promoted macrophage activation. This information may facilitate the development of potential therapeutic strategies for the prevention and treatment of inflammatory diseases, such as atherosclerosis.

Project description:AIM:The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion. METHODS AND RESULTS:By using immunoassaying and western blot analyses, we found that ?1 and ?2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of ?2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a ?-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased ?1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-?2-AdR levels while decreasing ?2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a. CONCLUSION:Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced ?2-AdR and increased ?1-AdR levels.

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Downregulation of angiopoietin-like protein 2 inhibits cementoblast differentiation partially by activating the ERK1/2 signaling pathway.

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