FIGURE 4.

p75NTR activation induces SREBP2 cleavage in a caspase-3-dependent manner. A, schematic structure of SREBP2 in the membrane, its interaction with the SCAP·INSIG complex, and the protease cleavage sites. In addition to the classical S1P and S2P proteases. there is a caspase-3 site at Asp-468 close to the membrane. Mutation of this site to alanine (D468A) was done as described under “Experimental Procedures.” B, 5 ng/ml pro-NGF increased the levels of cleaved caspase-3 (17 kDa) in a time-dependent manner in Huh7 cells. C, 50 ng/ml NGF increased caspase-3 cleavage that was blocked by using 50 μm Boc, a broad caspase inhibitor (left), and SREBP2 cleavage that was blocked by 1 μm of the specific caspase-3 inhibitor, Z-DEVD-fmk (DEVD). D, cells were transfected with the expression plasmids encoding DN-caspase-3 and further stimulated with 50 ng/ml NGF for 16 h. GFP-expressing plasmid was used as a control. Expression of DN-caspase-3 blocked the increase in cleaved SREBP2 induced by NGF. Left, immunoblot. Right, quantification. Values are mean ± S.E., n = 4. ***, p < 0.001 for NGF versus C, and for NGF + DN-Casp3 versus NGF. E, cells were transfected with mutant SREBP2-D468A (mut-SREBP2) expression plasmid for 24 h followed by stimulation with 5 ng/ml pro-NGF for 16 h. GFP-expressing plasmid was used as a control. Levels of LDLR were determined by immunoblotting. Left, immunoblots. Right, quantification. Values are mean ± S.E., n = 6. *, p < 0.05 for pro-NGF versus C in GFP-expressing cells. There was no increase in LDLRs in mut-DREBP2-expressing cells. The inset shows the levels of full-length (FL) SREBP2 in control and mut-SREBP2-expressing cells.