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The profound effects of transforming growth factor beta1 (TGF-beta1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-beta1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-beta1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-beta1 null mice (TGF-beta-1(-/-)). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-beta1(+/+)) and TGF-beta1 null mice were compared by cell counting, by 3H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-alpha1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-beta1(+/+) and TGF-beta1(-/-) embryonic fibroblasts. We report that TGF-beta1(-/-) cells proliferated at about twice the rate of TGF-beta1(+/+) cells. Further, TGF-beta1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-alpha1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-beta1(+/-) and TGF-beta1(-/-) cells could be eliminated by treatment of TGF-beta1(+/+) cells with a neutralizing antibody of TGF-beta1. Thus, our results are consistent with the hypothesis that TGF-beta1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts.

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells to Th1 cells; however, relatively few IL-12 target genes have been identified. To better clarify the molecular basis of IL-12 action, we set out to characterize genes up-regulated by IL-12, first by contrasting IL-12- and IFN-α-inducible genes. We identified several genes up-regulated by IL-12, namely, MIP-1α, MIP-1β, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription factor regulated by IFNs that is also essential for Th1 responses. We demonstrated that IL-12 directly up-regulates IRF-1 to the same extent as IFN-α in normal human T cells and in NK cells. We showed that IL-12 had a direct effect on IRF-1, an effect not mediated indirectly by the induction of IFN-γ production. Furthermore, IL-2 and IL-12 synergistically induced IRF-1, whereas IFN-α and IL-12 did not. The participation of STAT4 in the regulation of IRF-1 was demonstrated in two ways. First, STAT4 was required for the IL-12-dependent transactivation of an IRF-1 reporter construct, and second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In contrast to IL-12, no up-regulation of IRF-1 was found in IL-4-stimulated cells, and IL-4 did not block IL-12-dependent up-regulation of IRF-1. Therefore, IRF-1 may be an important contributor to IL-12 signaling, and we speculate that the defective IL-12 responses seen in IRF-1−/− mice might be attributable, in part, to the absence of this transcription factor.

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