Figure 4

Modulation of alkaline phosphatase, osteocalcin and collagen type 1 in OA Ob by inactivating leptin signaling. Confluent normal and OA Ob were treated for their last two days of culture with either media alone containing 0.5% FBS with or without 1,25(OH)₂D₃ (50 nM) as per indicated for the individual markers. Cells were treated with either exogenous leptin, antibodies against leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for 30 minutes prior to the addition of 1,25(OH)₂D₃ except for CICP that was performed in the absence of 1,25(OH)₂D₃. At the end of the 48 h incubation, the supernatant was kept for osteocalcin and for collagen production, and cells were lyzed in ALPase buffer prior to measuring alkaline phosphatase activity by substrate hydrolysis. A) Results of alkaline phosphatase activity for normal OB; B) Results of alkaline phosphatase activity for OA OB; C) Results of osteocalcin release by OA Ob; D) Results of CICP production; E) Confluent OA Ob were incubated in Ham's F12/DMEM media without serum and containing 1% ITS. Cells were treated with or without exogenous leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for their last 48 hours of culture. Results of TGF-β1 levels in supernatants are shown. The results are the mean ± SEM of n = 4 normal and n = 9 OA Ob preparations.