Dataset Information

Leptin inhibits AMPK?2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the expression of osteogenic markers.

ABSTRACT: AMP-activated protein kinase (AMPK) is of biological and clinical importance for regulating cellular and systemic energy homeostasis. Although AMPK?1, one of the two AMPK's catalytic subunit ?, expresses in the bone and stimulates bone nodule formation, the role of AMPK?2 in osteogenesis remains incompletely understood. The aim of this study was to determine the role of AMPK?2 in osteocytic MLO-Y4 cellproliferation and the expression of osteogenic markers. The current study silenced AMPK?2 in MLO-Y4 cells by transfection with pLKO.1-AMPK?2-shRNA vector and analyzed cell proliferation and the expression of osteogenic markers in MLO-Y4 cells with or without 100 ?g/ml leptin treatment through CCK-8, Real-time PCR, Western blot and RNA-seq assay. We found that knockdown of AMPK?2 significantly decreased the mRNA level of AMPK?2 and the cell proliferation of MLO-Y4 cellsas well as the mRNA and protein levels of OPG, OCN, OPN, ALP and BMP6 and the protein expression of p-Smad5/Smad5. However, leptin treatment increased the MLO-Y4 cell proliferation and the expression of these osteogenic markers in MLO-Y4 cells with or without AMPK?2 silencing. Furthermore, RNA-seq assay showed 1019 transcriptors decreased in AMPK?2-silencing group and 995 transcriptors increased in leptin group compared with control group, respectively. 737 transcriptors decreased in AMPK?2-silencing group and 1282 transcriptors increased inleptin group compared with AMPK?2-silencing+leptin group, respectively. These findings suggest that AMPK?2 knockdown inhibited MLO-Y4 cell proliferation and osteogenic marker expressions, which implicates an important role of AMPK?2 in osteogenesis in vitro.

SUBMITTER: Fan Q

PROVIDER: S-EPMC6965451 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Leptin inhibits AMPKα2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the expression of osteogenic markers.

Fan Qing Q Li Hao H Liu Zhu Z Zhang Zhiqiang Z Li Hai H Ding Jing J Zhang Ziming Z

International journal of clinical and experimental pathology 20170801 8

AMP-activated protein kinase (AMPK) is of biological and clinical importance for regulating cellular and systemic energy homeostasis. Although AMPKα1, one of the two AMPK's catalytic subunit α, expresses in the bone and stimulates bone nodule formation, the role of AMPKα2 in osteogenesis remains incompletely understood. The aim of this study was to determine the role of AMPKα2 in osteocytic MLO-Y4 cellproliferation and the expression of osteogenic markers. The current study silenced AMPKα2 in ML ...[more]

PMID: 31966708

Similar Datasets

Project description:Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among the proteins with elevated abundances in dendrites were molecules that regulate cytoskeletal function, cell motility and membrane adhesion. Our combined transcriptomic/proteomic analysis of the response of MLO-Y4 osteocytic cells to LPA indicates that dendritogenesis is a membrane- and cytoskeleton-driven process with actin dynamics playing a particularly critical role.

Project description:Osteocyte plays an essential role in bone metabolism by regulating osteoblast and osteoclast activities. Dysfunction or apoptosis of osteocyte will severely endanger the bone homeostasis and result in bone diseases such as osteoporosis. Osteoporosis has been considered as one of the diabetes complications; however, the mechanism is still to be discovered. Advanced glycation end products (AGEs), as the main pathogenic factor of diabetes mellitus, have the capacity to induce osteocyte apoptosis thus sabotaging bone homeostasis. Here, we examined the role of AGE during osteocyte apoptosis and how this effect would affect osteocyte's regulation of osteoblast and osteoclast. Mouse osteocyte-like MLO-Y4 cells were used to study the properties of osteocyte and to examine its biological and pathological function. MTT assay and Annexin V assay showed that AGE significantly induce MLO-Y4 cell apoptosis. qPCR and Western blot results have shown that AGE upregulates proapoptotic gene p53 and its downstream target gene Bax, which leads to enhanced activation of caspase-3, thus inducing apoptosis in MLO-Y4 cells. Increased expression of sclerostin and RANKL in osteocytes has shown that AGE induces osteocyte dysfunction thus severely damaging the bone homeostasis by decreasing osteoblast and increasing osteoclast activities. Furthermore, the role of the transcription factor FOXO1, which is intensely associated with apoptosis, has been determined. Western blot has shown that AGE significantly decreases Akt activities. Immunofluorescence has shown that AGE promotes FOXO1 nuclei localization and enhances FOXO1 expression. Silencing of FOXO1 suppressed AGE-enhanced apoptosis; mRNA and protein expressions of cleaved caspase-3, sclerostin, and RANKL were downregulated as well. Moreover, exogenous FOXO1 increased caspase-3 mRNA levels and caspase-3 transcriptional activity. Lastly, ChIP assay has established the capacity of FOXO1 binding directly on the caspase-3, sclerostin, and RANKL promoter region in AGE environment, providing the mechanism of the AGE-induced osteocyte apoptosis and dysfunction. Our results have shown that FOXO1 plays a crucial role in AGE-induced osteocyte dysfunction and apoptosis through its regulation of caspase-3, sclerostin, and RANKL. This study provides new insight into diabetes-enhanced risk of osteoporosis given the critical role of AGE in the pathogenesis of diabetes and the essential part of osteocyte in bone metabolism.

Project description:We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/β-Catenin pathway.

Dataset Information

Leptin inhibits AMPK?2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the expression of osteogenic markers.

Publications

Leptin inhibits AMPKα2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the expression of osteogenic markers.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets