Hyaluronan Promotes Expression Of α5-Integrin Subunit In Cultured Chondrocytes

PrefaceIntegrins are a large family of glycoprotein receptors that mediate interactions between cell - cell and cell - matrix. Integrins are heterodimeric transmembrane proteins consisting of an α and an β subunit. both subunits have large extracellular domains that interact with extra cellular matrix proteins and relatively small cytoplasmic domains that interact with cytoskeletal proteins. Integrins binding of ECM ligands results in the formation of signaling complexes which play a key role in the regulation of cell survival, proliferateion, differentiation, and matrix remodeling. The α5β1 integrin is the prominent chondrocyte integrin which is very important for the survival of chondrocytes. Hyaluronan is used in the treatment of OA by intraarticular injection. Several studies noted that hyaluronan could protect the articular during the progression of OA. The present study investigated the in vitro effect of hyaluronan on the expression of α5 - integrin subunit to further clarify the mechamism of protection of hyaluronan on chondrocyte.Materials and MethodsChondrocytes were obtained by enzymatic digestion of minced fragments of cartilage. The isolated chondrocytes were cultured on coverslips. Immunohisto-chemical assessment of chondrocytes phenotype was performed using anti - type Ⅱ collagen antibody. After the cultured chondrocytes treated with hyaluronanfor an hour, Immunocellularchemical assessment of expression of chondrocytes α5 - integrin subunit was performed using anti -α5 - integrin subunit monoclonal antibody. Obtained photograph was measured using micro - image analyzer software.ResultsThe assessment of articular chondrocytes: All of the articular chondrocytes used in the following experiments were positive for type Ⅱ collagen, type Ⅱ collagen locates in extra cellularmatrix, and the positive cell plasma are stained to be yellow or brown.The effect of hyaluronan on the expression of chondrocytes α5 - integrin subunit: All of the chondrocytes were positive for a5 - integrin subunit. α5 -integrin subunit locates in cell plasma/membrane and the positive cell plasma/ membrane are stained to be yellow or brown. The expression level of experimental and control group was 137. 61 ± 6. 36 and 100. 84 ± 3. 45. The difference was significant (p<0.01).DiscusionThe stable microenviroment of the chondrocytes is very important for the functions of the cells and the maintains of the articular cartilage tissue composition. Chondrocytes receive information from the external environment via three potential sources: mechanical signals, soluble mediators, and interactions with the ECM.Chondrocyte attachment to the ECM appears to be mediated by integrins, After binding to the ECM, integrins bind to cytoskeltal elements and promote cy-toskeletal reorganization. Moreover, cytoskeletal alterations in response to a specific integrin - ligand combination may result in increased or decreased gene expression. The α5β1 integrin play a key role in the mediation of cell survival signal transmition and the adhesion function of the chondrocyte. Expression of the integrin decreases in many cell types during differentiation and develop-ment and increase during wound healing. Judit et al reported that the isolated chondrocytes died after treated with a monoclonal blocking antibody to the α5 -integrin subunit and the chondrocyte death associated with apoptosis; Treatment of chondrocytes with blocking antibodies for other integrin subunits do not cause significant chondrocyte death. These results demonstrate that the α5β1 integrin provides survival signals for normal and osteoarthritic human articular chondrocytes in vitro. In the pathogenetic pathway of OA, chondrocyte apoptosis is now considered a major event, since apoptotic bodies may remain in the cartilage ECM due to the absence of phagocytes and may accelerate matrix degradation by releasing degrdative enzymes. The loss of chondrocyte - matrix interactions cause a reduction in the main survival signal, so that apoptotic death may increase.Hyaluronan has been used in OA therapy for several years, but a clear comprehension of its mechanism of action is still lacking, and some conflicting results have been published on its clinical effects. Besides anti - inflammatory effects and a lubricative agent, several studies reported that hyaluronan has inhibitory effects on chondrocyte apoptosis. Kenji et al reported that the apoptotic chondrocytes was significantly lower after intrarticulr injection of hyaluronan in experimentally induced OA, the results suggest hyaluronan protect against chondrocyte apoptosis during the development of OA. Gina reported that hyaluronan exerted an antiapoptotic effect on anti - Fas - induced chondrocyte apoptosis. In fact, proteoglycan breakdown of the cartilage matrix leads not only to the progressive erosion of the cartilage surface, but also to the release into the synovial fluid of ECM fragments which have been proven to induce chondrocyte apoptosis. Hashimoto et al demonstred a strict correlation between proteoglycan depletion and chondrocyte apoptosis in OA. Shimazu et al reported that hyaluronan (at molecular weights ranging from 500 kd to 1,900 kd) is a potent inhibitor of proteoglycan depletion in rabbit articular cartilage. Supporting the hypothesis that hyaluronan may contribute to the restoration of homeostasis in cartilage ECM.In chondrocytes, the only known partner for α5 is β1 subunit. In the present study, hyaluronan promotes the expression of the α5 - integrin subunit in...