FIG. 1.

Modulation of Raf-1-induced MMP-1 promoter activity by stress-activated MAPK pathways. (A to C) After transfection and glycerol shock, the cultures were maintained for 36 h in 1% FCS, and CAT activity was measured from cell lysates. Transfection efficiency was monitored by cotransfecting the cells with 4 μg of RSV–β-galactoside construct. The values represent the mean of at least two independent experiments, each performed in duplicate. (A) NIH 3T3 fibroblasts were transiently transfected with indicated amounts of the expression vector for the constitutively active form of Raf-1 (RafBXB) together with the -2278CLCAT construct (2 μg), containing 2,278 bp of the human MMP-1 gene promoter linked to the CAT gene. (B) NIH 3T3 fibroblasts were transiently transfected with increasing amounts (0.5, 1, 2, and 4 μg) of expression vectors for constitutively active MKK3 [MKK3(E)], MKK6 [MKK6(EE)], TAK1 (ΔNTAK1), and Rac (RacQL), or with Raf-1 (RafBXB) (2 μg) in combination with MMP-1 promoter-CAT construct -2278CLCAT (2 μg). (C) NIH 3T3 fibroblasts were transiently transfected with MMP-1 promoter-CAT construct -2278CLCAT (1 μg) with the expression vector for constitutively active Raf-1 (RafBXB) alone or in combination with expression vectors for constitutively active MKK3, MKK6, and TAK1 (1 μg each). (D) NIH 3T3 fibroblasts were transiently transfected with 4 μg of expression vectors for the constitutively active form of TAK1 (ΔNTAK1) and Rac [Rac(QL)] together with the expression vector for Flag-tagged p38α. Activity of p38α immunoprecipitated from cell lysates with anti-Flag antibody was determined with GST–ATF-2 as substrate. The levels of p38α in cell lysates were determined by Western blot analysis with anti-Flag antibody. (E) NIH 3T3 fibroblasts were transiently transfected with expression vectors for the constitutively active form of TAK1 (ΔNTAK1) (2 μg), MKK3b [MKK3b(E)] and MKK6b [MKK6b(E)] (4 μg), together with the expression vector for constitutively active Raf-1 (RafBxB) (2 μg). The level of RafBxB in cell lysates was determined by Western blot analysis with c-Raf-1 antibody. (F) NIH 3T3 fibroblasts were transiently transfected with 4 μg of expression vectors for constitutively active forms of MKK3b [MKK3b(E)] and MKK6b [MKK6b(E)], together with expression vectors for wild-type p38 isoforms (p38α, -β, -γ, and -δ) (4 μg each). Control cultures were transfected with the corresponding empty expression vector. Cultures were then maintained for 36 h in 1% FCS–DMEM, and the activity of p38 isoforms immunoprecipitated from cell lysates with anti-Flag antibody was determined using GST–ATF-2 as substrate. CTL, empty expression vector.