Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape.

Benya, Paul D.; Brown, Peter de Nully; Padilla, Silvia R.

doi:10.1083/jcb.106.1.161

Cited by 179 publications

(105 citation statements)

References 54 publications

(87 reference statements)

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“…Indeed, the microfilament-disruptive drug, cytochalasin, induces reexpression of chondrocyte phenotype in monolayers of both subcultured and retinoic acid-modulated chondrocytes (38,39). Although microfilament modification is an important mediator of the chondrocyte phenotype, ERK regulation of chondrocyte dedifferentiation is not mediated by the modification of actin cytoskeleton.…”

Section: Discussionmentioning

confidence: 99%

Maintenance of Differentiated Phenotype of Articular Chondrocytes by Protein Kinase C and Extracellular Signal-regulated Protein Kinase

Yoon¹,

Kim²,

Oh³

et al. 2002

Journal of Biological Chemistry

150

165

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The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signalregulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC␣ was essentially the same as that of type II collagen during de-and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC␣, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC␣ in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC␣ did not affect type II collagen levels, suggesting that PKC␣ expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by downregulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of Ncadherin, ␣-catenin, and ␤-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.

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Section: Discussionmentioning

confidence: 99%

Maintenance of Differentiated Phenotype of Articular Chondrocytes by Protein Kinase C and Extracellular Signal-regulated Protein Kinase

Yoon¹,

Kim²,

Oh³

et al. 2002

Journal of Biological Chemistry

150

165

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“…Forcing spherical cell shape by pharmacological inhibition of actin polymerization is sufficient to stimulate dedifferentiated cells to re-express chondrogenic matrix molecules (21)(22)(23)(24) and promotes mesenchymal cells to commit to the chondrogenic lineage (24,25). We therefore speculated that signaling pathways that regulate cell shape also regulate chondrocyte differentiation.…”

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confidence: 99%

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Woods¹,

Wang

Dupuis

et al. 2007

Journal of Biological Chemistry

109

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The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.Chondrocytes are the cellular component of cartilage, responsible for generating and maintaining its extracellular environment (1). Cartilage has multiple functions such as providing a cushion on the articular surfaces of joints (2), providing a template for the formation of endochondral bone (3), and contributing to fracture repair (4). The formation of cartilage templates in endochondral ossification begins with condensation of mesenchymal cells, increased expression of the cell adhesion molecules N-cadherin and N-CAM, and therefore increased cell-cell interactions (5-7). As cells become chondrogenic, the expression of these adhesion molecules is decreased (8). Cells within these condensations commit to the chondrogenic lineage, acquire a spherical cell morphology and induce expression of the essential chondrogenic transcription factor Sox9 (9, 10). Sox5 and Sox6 cooperate with Sox9 to control chondrogenesis and are themselves under the transcriptional control of Sox9 (9,11,12). Together, these transcription factors activate transcription of the major chondrogenic matrix genes, collagen II and aggrecan (11,(13)(14)(15). Furthermore, increased glycosaminoglycan (GAG) 3 content is another marker of the chondrogenic extracellular matrix (1). The production of glycosaminoglycans are partially regulated by the enzymes chondroitin 4-sulfotransferase 11 (Chst 11) and chondroitin 6-sulfotransferase 3 (Chst 3), which have been determined to be important for proper cartilage and bone formation (16,17).Although many...

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“…However, recent in situ hybridization studies of cultured chick embryo chondrocytes indicated a lack of correlation between changes in cell shape and expression of cartilage-specific genes (20). Other investigators have related the modulation of cartilage-specific gene expression in cultured chondrocytes to the degree of polymerization of the actin cytoskeletal elements (21,22).…”

mentioning

confidence: 99%

Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

Reginato

Iozzo

Jimenez

1994

Arthritis & Rheumatism

105

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Objective. To establish long-term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage-specific phenotype.Methods. Chondrocytes isolated from 20-24-week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly-(2-hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, 11, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human-specific complementary DNAs.Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosynthetic studies demonstrated the maintenance of a Cartilagespecific phenotype throughout the 180 days of culture, Submitted for publication August 31, 1993; accepted in revised form March 20, 1994. with the production of aggrecan and types 11, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type I1 procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture.

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Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape.

Cited by 179 publications

References 54 publications

Maintenance of Differentiated Phenotype of Articular Chondrocytes by Protein Kinase C and Extracellular Signal-regulated Protein Kinase

Maintenance of Differentiated Phenotype of Articular Chondrocytes by Protein Kinase C and Extracellular Signal-regulated Protein Kinase

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

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