FIG. 2.

PDGF induction of MCP-1 involves interactions between the heptamer TTTTGTA and a 5′ I* element. (A) At the top is a schematic of the structures of six tagged MCP-1 reporter constructs. Constructs 1 through 4 and 6 contain 104 bp of 3′ untranslated sequences (that include the polyadenylation signal but not the heptamer). Only construct 5 contains the heptamer within its 3′ untranslated sequences. The details of the MCP-1 schematics are otherwise as described in the legend to Fig. 1. Constructs 1 and 2 in this figure are identical to constructs 1 and 5, respectively, in Fig. 1. Construct 3 is derived from construct 2 by readdition of the 707-bp EcoRI-AocI fragment. Construct 4 is derived from construct 2 by readdition of the 695-bp SpeI-EcoRI fragment. Construct 5 is derived from construct 4 by readdition of the heptamer to the proximal 3′ untranslated sequences. Construct 6 is derived from construct 4 by readdition of the heptamer to the distal 5′ flanking sequences. The PDGF-regulated 5′ I* elements are indicated. The PDGF inducibilities of the six constructs in transfection experiments are summarized on the right. At the middle are RNase protection assays of 40 μg of total cellular RNA that was prepared from NIH 3T3 fibroblasts transiently transfected with 5 μg of the constructs shown, allowed to become quiescent, and then not exposed (−) or exposed (+) to the B-B isoform of PDGF (30 ng/ml) for 3 h. The numbers refer to the tagged MCP-1 constructs diagrammed at the top. The 305- and 241-nt protected fragments corresponding to expression of the transfected and tagged (T) or endogenous (E) MCP-1 genes, respectively, are indicated. The experiment was performed four times with similar results. The PDGF inductions obtained with constructs 3 and 4 were 10 to 64 and 10 to 15%, respectively, of those obtained with construct 2 in these transfections. The PDGF inductions obtained with construct 5 were 2.1 to 9.7 times greater than those obtained with construct 4 in these transfections. The PDGF induction decreases observed with constructs 3 and 4, compared to construct 2, are both statistically significant (P < 0.05 by the Wilcoxon two-sample test). The PDGF induction increases observed with construct 5, compared to construct 4, are significant (P < 0.05 by the Wilcoxon two-sample test). At the bottom are RNase protection assays of 15 μg of total cellular RNA taken from the transfections shown above and analyzed with an alpha-globin riboprobe. (B) At the top are RNase protection assays of 40 μg of total cellular RNA that was prepared from NIH 3T3 fibroblasts transiently transfected with 4 μg of the constructs shown, allowed to become quiescent, and then not exposed (−) or exposed (+) to the B-B isoform of PDGF (30 ng/ml) for 3 h. The numbers refer to the tagged MCP-1 constructs described at the top, except for constructs 5m1, 5m2, and 5m3. The latter constructs are derived from construct 4 by addition of the mutant heptamers TTTTATG, GGGGGTA, and TTTTGGA, respectively, to the 3′ untranslated sequences. Mutations of the wild-type heptamer sequence are underlined. The 305- and 241-nt protected fragments corresponding to expression of the transfected and tagged (T) and endogenous (E) MCP-1 genes, respectively, are indicated. The experiment was performed four times with similar results. The decreased PDGF inductions obtained with constructs 5m1, 5m2, and 5m3 varied from 6 to 15%, 8 to 25%, and 50 to 60% of those obtained with construct 5, respectively, in these transfections. The PDGF induction differences obtained with constructs 5m1, 5m2, and 5m3, compared to construct 5, are all statistically significant (P < 0.05 by the Wilcoxon two-sample test). The PDGF induction increases observed with constructs 5 and 6, compared to construct 4, are statistically significant (P < 0.05 by the Wilcoxon two-sample test). At the bottom are RNase protection assays of 15 μg of total cellular RNA taken from the transfections shown above and analyzed with an alpha-globin riboprobe.