Expression of metalloproteinases and their inhibitors change when sternal cartilage is organ cultured with integrin antibodies.
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Prior to bone deposition, chondrocytes differentiate into hypertrophie chondrocytes and change gene expression from collagen types Q, DC and XI to X. Cartilage is replaced by bone as blood vessels invade and collagen type I is deposited. Cartilage derived metalloproteinase inhibitors (TIMPs) contribute to the avascularity of cartilage. We have previously shown that chondrocyte differentiation is inhibited when whole chick sterna are organ cultured in the presence of specific integrin antibodies (anti-l, -o2. -a3), suggesting a role in differentiation. Blocked differentiation was determined by decreased sternal growth, lack of type X collagen secretion and decreased cell size. We now ask if integrin receptor signaling is important for regulating the production of matrix metalloproteinases (MMPs) and TIMPs. Whole embryonic day 14 sterna were incubated at 37C for 8 days in serum free culture media in the absence or presence of integrin antibodies and control IgG antibodies. Conditioned media was collected daily and proteins were extracted from whole sterna. Western blot analysis with the TIMP, anti-cartilage derived inhibitor (GDI), revealed molecular weight bands at approximately 35,27 and 18 Kd, representing GDI, chondrocytes derived inhibitor (ChDI) and possibly TIMP-3, respectively. The amount of inhibitor increased when sterna were cultured in the presence of integrin antibodies. Zymography revealed different amounts of gelatinase (94, 72 and 66 Kd) at different developmental stages. These results suggest that cell-matrix interactions via integrin receptors are necessary for sternal growth, chondrocyte differentiation and regulation of MMPs and TIMPs.
