| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
|
|---|
| Research Abstract |
KIT is essential for melanocyte development, but the factors which induce KIT expression in melanoblasts have not been well identified. To clarify the mechanisms of the KIT expression, we used a KIT-positive, and a KIT-negative melanoblast cell line, NCCmelb4 and NCCmelb4M5, respectively. Those were established from neural crest cells in our laboratory. KIT mRNA by RT-PCR was detected in NCCmelb4M5 cells but KIT protein was not detected. However, only a few KIT positive cells (about 0.1%) were detected by immunostaining. When 12-o-tetradecanoyl-13-acetate (TPA) and cholera toxin (CT) were added to the culture medium, NCCmelb4M5 became weakly positive to KIT by immunostaining. The optimum concentration of TPA was 50 ng/ml and that of CT was 1 nM. At 72 hours after the addition of TPA and CT, the expression level of KIT was maximum by western blotting and 21% of NCCmelb4M5 cells became positive to KIT by FACS analysis. α-MSH (100 nM) and DBcAMP (0.5 mM) weakly induced the KIT expression of NCCmelb4M5 as shown by immunostaining, which was not detected by western blotting. SCF (50 ng/ml), endothelin 3 (100 nM), TGF-β (10 ng/ml) had no effect on the KIT expression as shown by immunostaining. Immunostaining revealed that when NCCmelb4 cells were treated with calphostin C (20 nM), a PKC inhibitor, and H-89 (2 μM), a PKA inhibitor, their KIT expression was decreased. FACS analysis showed a 26% decrease of KIT positive cells in NCCmelb4 at 72 hours after the addition of calphostin C and H-89. These results indicate that PKC or PKA is partially involved in the regulation of KIT expression of melanocyte precursors.
|
