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TGFβ REGULATION OF ANKH (PROGRESSIVE ANKYLOSIS) EXPRESSION IN ATDC5 CELLS. PA Oca, CJ Williams Department of Medicine, Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA.
The following studies were performed to evaluate the expression of ANKH in response to TGFß during chondrogenesis in ATDC5 cells, an in vitro model of growth plate differentiation. Cells subjected to differentiation with and without TGFß and the progression of differentiation was monitored using markers of chondrogenesis, such as expression of Col10 and Apk2 (alkaline phosphtase). Inhibitors of TGFß signaling were used to identify the signaling pathway employed in TGFß regulation of ANKH expression, specifically Go6976 which is a traditional PKC inhibitor that requires Ca+2 to function. Finally, inhibition of the L type calcium channel alpha 1c (Lt1c) was studied to determine its role in the regulation of ANKH expression in response to TGFß. TGFß produced an increase in ANKH expression at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. To determine the TGFß-activated signaling pathway, cells with and without TGFß at day 4 (immature), day 14 and day 32 were incubated in the presence of specific inhibitors. The TGFß treatment activated the Ca+2-dependent protein kinase C (PKC) signaling pathway. Since previous reports of chondrogenesis implicated Ltcs in Ca+2-dependent PKC signaling, we explored the activity of the Lt1c in the TGFß-stimulated ANKH response in ATDC5 cells. Our results indicated that inhibition of Lt1c with nifedipine inhibited the TGFß stimulated increase in ANKH expression at all phases of differentiation. It was also observed that Lt1c expression remained unchanged during chondrogenesis both with and with out TGFß. Expression of ANKH during chondrogenesis demonstrates a bimodal response to TGFß during the proliferative and hypertrophic phases of differentiation. The TGFß response is mediated by the Ca+2-dependent PKC signaling pathway and requires influx of calcium via high voltage L type calcium channel alpha 1C.
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