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Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture
Author(s) -
Parreno Justin,
Nabavi Niaki Mortah,
Andrejevic Katarina,
Jiang Amy,
Wu Pohan,
Kandel Rita A.
Publication year - 2017
Publication title -
journal of anatomy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 118
eISSN - 1469-7580
pISSN - 0021-8782
DOI - 10.1111/joa.12554
Subject(s) - chondrocyte , aggrecan , tubulin , microbiology and biotechnology , actin , cytoskeleton , biology , microtubule , chemistry , cell , biochemistry , in vitro , medicine , pathology , alternative medicine , osteoarthritis , articular cartilage
Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non‐chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly ( P < 0.05) increased gene expression of proliferation‐associated molecules (cyclin D1 and ki67), as well as significantly ( P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast‐like matrix, type I collagen ( COL 1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly ( P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL 1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL 1 gene expression. To confirm that actin status regulated COL 1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL 1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL 1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL 1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.