Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression.

Abstract

Abstract. The differentiated phenotype of rabbit articular chondrocytes was modulated in primary culture by treatment with 1 pg/ml retinoic acid (RA) and reexpressed in secondary culture by treatment with the microfilament-disruptive drug dihydrocytochalasin B (DHCB) in the absence of RA. Because the effective dose of DHCB (3 ~tM) did not elicit detectable cell rounding or retraction, the nature and extent of microfilament modification responsible for induction of reexpression was evaluated. The network of microfilament stress fibers detected with rhodamine-labeled phalloidin in primary control chondrocytes was altered by RA to a "cobblestone" pattern of circularly oriented fibers at the cell periphery. Subsequent treatment with DHCB resulted in rapid changes in this pattern before overt reexpression. Stress fibers decreased in number and were reoriented. Parallel arrays of long fibers that traversed the cell were evident, in addition to fiber fragments and focal condensations of staining. Immunofluorescent staining of intermediate filaments revealed a marked decrease in complexity and intensity during RA treatment but no change during reexpression. An extended microtubular architecture was present throughout the study. These results clearly identify microfilaments as the principal affected cytoskeletal element and demonstrate that their modification, rather than complete disruption, is sufficient for reexpression. The specificity of DHCB and the reorientation of these filaments before reexpression of the differentiated phenotype suggests a causative role in the mechanism of reexpression. CHONDROCYTES exhibit high levels of synthesis of cartilage-specific proteoglycan and type II collagen as a major part of their differentiated phenotype. The expression of this phenotype has been shown to be responsive to changes in cell shape, the maintenance (13) or resumption (5) of spherical conformation promoting the expression of the differentiated phenotype. In contrast modulating agents such as fibronectin (17, 26) and retinoic acid (RA) t (4), which appear to enhance both adhesion and spreading of cultured chondrocytes, also enhance the rate at which the differentiated phenotype is lost. These observations, together with the results of more recent studies on the effects of microfilament-modifying drugs on chondrogenesis (18, 27), suggest the involvement of some element of the cytoskeleton in modulation of the chondrocyte phenotype.To study this involvement, the cytoskeletal architecture of rabbit articular chondrocytes has been examined by immunofluorescent techniques during both RA modulation of the differentiated phenotype and its subsequent reexpression as induced by the microfilament-modifying drug dihydrocytochalasin B (DHCB). Transitions in the synthesis of genetically distinct collagen types were used as markers for defined phenotypic change, and are reported separately (4, 6). Briefly, RA modulation of the phenotype was marked by a cessation of type II collagen synthesis and a transient stimulation of type III a...

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αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

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Rac1/Cdc42 and RhoA GTPases Antagonistically Regulate Chondrocyte Proliferation, Hypertrophy, and Apoptosis

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