FIG. 6.

IFN-β mRNA induction by poly(I-C) depends on Smad3 expression. Induction of IFN-β mRNA was monitored by Northern hybridization. (A) Poly(I-C) treatment of MEFs induced expression of IRF-7 mRNA, as assessed by RT-PCR at the indicated times following initiation of treatment. (B) Effect of poly(I-C) on IRF-7 labeled with [³²P]orthophosphate in vivo. Poly(I-C) induced a mobility shift of ³²P-labeled IRF-7, as assessed after 4 h of treatment, similar to what is observed in response to viral infection. Similar results were also observed after 2 h of treatment with poly(I-C) (data not shown). (C) Effect of poly(I-C) on the transcriptional activity of IRF-7 at the p31x2-Luc promoter/reporter. Poly(I-C) enhanced the transcriptional activity of IRF-7 at different expression levels in transfected MEFs. The poly(I-C)-induced increase of transcription in vector control MEFs was likely a reflection of endogenous IRF-3 and IRF-7 expression (A). (D) Smad3^−/− and matched normal MEFs were treated with poly(I-C), and RNA was isolated at the indicated times following addition of poly(I-C). The absence of Smad3 delayed and decreased early-phase IFN-β induction. (E) Expression of Smad3 in Smad3^−/− cells, infected with the LPCX-Smad3 vector, conferred earlier induction of endogenous IFN-β expression in response to poly(I-C) treatment. LPCX-infected control cells and Smad3-expressing cells were treated with poly(I-C), and total RNA was isolated at the indicated times after treatment. The top portions of panels D and E show IFN-β mRNA under the same exposure condition on the same X-ray film, while the lower portions of the panels show the ethidium bromide-stained gels. (F) Expression of Smad3 in Smad3^−/− cells infected with the retroviral LPCX-Smad3 vector, as shown by Western blotting to detect Flag-tagged Smad3.