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The MEK-ERK1/2 signaling pathway regulates hyaline cartilage formation and the redifferentiation of dedifferentiated chondrocytes in vitro.

ABSTRACT: The aim of this study was to investigate the role of the mitogen-activated protein kinase kinase-extracellular signal-regulated kinases 1/2 (MEK-ERK1/2) signaling pathway in chondrocyte differentiation and cartilage tissue construction in vitro. Chondrocytes were stimulated with rat serum (RS) and fetal bovine serum (FBS), and chondrocyte phenotypes were investigated microscopically. Chondrocyte proliferation was analyzed using fluorescence activated cell sorting (FACS) and the CCK8 method. Protein and mRNA expressions were assessed by western blot and RT-qPCR. Constructed cartilage tissues were examined by Safranin O-Fast Green FCF staining and immunofluorescence. In contrast to FBS, RS induced rapid dedifferentiation of chondrocytes and decreased type II collagen expression and proteoglycan synthesis. ERK1/2 and type I collagen expression increased during dedifferentiation and decreased during redifferentiation. Increased MEK-ERK1/2 pathway activity resulted in chondrocyte dedifferentiation, and inhibition of ERK1/2 by the inhibitor PD0325901 reversed dedifferentiation and led to redifferentiation. These data suggest strongly that inhibition of MEK-ERK1/2 activation prevents chondrocyte dedifferentiation and fibrocartilage formation.

SUBMITTER: Wang X

PROVIDER: S-EPMC6220221 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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The MEK-ERK1/2 signaling pathway regulates hyaline cartilage formation and the redifferentiation of dedifferentiated chondrocytes in vitro.

Wang Xuezong X Xue Yan Y Ye Weiwei W Pang Jian J Liu Zhenfen Z Cao Yuelong Y Zheng Yuxin Y Ding Daofang D

American journal of translational research 20181015 10

The aim of this study was to investigate the role of the mitogen-activated protein kinase kinase-extracellular signal-regulated kinases 1/2 (MEK-ERK1/2) signaling pathway in chondrocyte differentiation and cartilage tissue construction <i>in vitro</i>. Chondrocytes were stimulated with rat serum (RS) and fetal bovine serum (FBS), and chondrocyte phenotypes were investigated microscopically. Chondrocyte proliferation was analyzed using fluorescence activated cell sorting (FACS) and the CCK8 metho ...[more]

PMID: 30416651

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Project description:Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (20% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9 days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72 h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic fibroblast growth factor, G-CSF, and PDGF were significantly higher in PRP than in HAS (p < 0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9 days significantly (p < 0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p < 0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p < 0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72 h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72 h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.

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The MEK-ERK1/2 signaling pathway regulates hyaline cartilage formation and the redifferentiation of dedifferentiated chondrocytes in vitro.

Publications

The MEK-ERK1/2 signaling pathway regulates hyaline cartilage formation and the redifferentiation of dedifferentiated chondrocytes in vitro.

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