Fibronectin (FN), an alternately spliced adhesion protein involved in development, healing, and neoplasia, accumulates in osteoarthritic (OA) cartilage at sites of greatest disease activity. As an initial step to determine which chondrocyte receptors are involved in recognition of the alternatively spliced V segment of FN, we performed cell surface labeling on RCJ nontransformed rat chondrocytes, followed by immunoprecipitation with antibodies to integrin subunits. α3, α5, αv, β1, β3, and β5, but not α4, subunits were detected. Subunits with electrophoretic mobilities consistent with αv and β3 were observed to coprecipitate. Next, we tested the capacity of function-blocking antibodies to integrin subunits (Abs) (each at 12.5 ug/ml) to inhibit adhesion by RCJ cells to plastic substrates coated with a glutathione S-transferase (GST) fusion protein bearing the V segment of rat FN (V-GST). Cells to be tested had been grown either on a substrate of collagen/agarose to promote differentiated (diff) chondrocyte gene expression, or on plastic to promote loss of diff gene expression. The adhesion achieved in the presence of Abs, expressed as a percentage of the value achieved by control cells adhering in the absence of Abs, is shown below. Data are expressed as the average standard deviation for six microtiter wells (two triplicate series of wells from two experiments). Based upon these results, we conclude that RCJ chondrocytes use αvβ3 integrin to recognize the alternatively spliced V segment of rat FN. Also, it appears that Ab inhibition of chondrocyte integrin recognition of the V segment may occur less readily in diff cells. Blocking Ab to: Plastic Collagen/Agarose α5 104.7+/-4.7 105.8+/-13.4 αv 5.7+/-7.3 23.5+/-17.3 α5+αv 1.2+/-0.4 10.6+/-4.8 β1 96.8+/-15.7 113.2+/-19.9 β3 11.8+/-12.6 42.3+/-25.3 β1+β3 7.5+/-7.2 19.9+/-16.5.
|Journal||Journal of Investigative Medicine|
|State||Published - Feb 1999|