Figure 5.

Ets-1 is required for the transforming growth factor (TGF)β induction of CCN2. (a) Dominant negative Ets-1 blocks the TGFβ induction of the CCN2 promoter. A CCN2 promoter/reporter construct driven by nucleotides -805 to +17 of the CCN2 promoter was transfected into fibroblasts along with empty expression vector or expression vector encoding dominant negative Ets-1 or Smad7, as indicated. Following serum starvation for 24 hours, cells were incubated in the presence or absence of 4 ng/ml TGFβ1 for 24 h, as indicated. Average ± standard deviation (N = 6) is shown (*p < 0.05). (b) Small interfering RNA (siRNA) recognizing Ets-1 mRNA suppresses the TGFβ induction of CCN2. Western blot analysis; fibroblasts were transfected either with control siRNA or siRNA recognizing Ets-1 or Fli-1 mRNAs. Following a serum starvation step of 24 h, cells were incubated in the presence or absence of a 4 ng/ml TGFβ1 for 24 h, as indicated. Proteins were blotted onto nitrocellulose, Membranes were probed with anti-Ets-1, anti-Fli-1 or anti-CCN2 antibodies, as indicated. Values below CCN2 western blot indicate relative amounts of CCN2 protein as determined by densitometry relative to actin. (c) siRNA recognizing Ets-1 mRNA suppresses the TGFβ-induction of CCN2. Immunofluorescence analysis; fibroblasts were transfected either with control siRNA or siRNA recognizing Ets-1. After a serum starvation step of 24 h, cells were incubated in the presence or absence of 4 ng/ml TGFβ1 for 24 h, as indicated. Cells were then fixed in paraformaldehyde, and CCN2 was detected with an anti-CCN2 antibody followed by incubation with an appropriate Texas Red-conjugated secondary antibody (red). Cells were costained with DAPI to detect nuclei (blue).