The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor beta (TGF-beta) superfamily of ligands. To discern the in vivo functions of one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost the ability to form Smad-containing DNA binding complexes and are unable to induce transcription from the TGF-beta-responsive promoter construct, p3TP-lux. Using the primary dermal fibroblasts, we also demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that Smad3 null MEFs are partially resistant to TGF-beta's antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-beta-mediated growth inhibition. We next examined cells in which Smad3 is most highly expressed, specifically cells of immune origin. Although no specific developmental defect was detected in the immune system of the Smad3 null mice, a functional defect was observed in the ability of TGF-beta to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-beta-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-beta and implicate Smad3 as a potential effector for TGF-beta in modulating immune system function.

. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice.

Members of the Smad family of proteins are thought to play important roles in transforming growth factor ␤ (TGF-␤)-mediated signal transduction. In response to TGF-␤, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-␤ on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-␤-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-␤-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-␤-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-␤. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.

Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNAbinding sites in response to transforming growth factor ␤ (TGF␤) and activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites that do not interact with Smads directly. Here, we report that Smad3 and Smad4 can physically interact with AP-1 family members. In vitro binding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. We show that Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGF␤. Providing evidence for the importance of this interaction between Smad and Jun proteins, we demonstrate that Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. Together, these data suggest that TGF␤-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors.Transforming growth factor ␤ (TGF␤) is a multipotent cytokine that regulates a variety of cellular activities, such as cell proliferation, differentiation, and extracellular matrix (ECM) formation. The combined actions of these cellular responses are likely to mediate more global effects of TGF␤ including its role in development, wound healing, immune responses, and the pathogenesis of cancer (1-3). The identification of genes transcriptionally regulated by TGF␤ and the elucidation of the molecular mechanisms responsible for this transcriptional regulation will help define how TGF␤ exerts its cellular effects and its role in resulting physiological processes. Although progress has been made in the identification of TGF␤ target genes, including the cyclin-dependent kinase inhibitors p21 and p15 (1, 2) and the ECM component plasminogen activator inhibitor-1 (PAI-1) (3), which has subsequently contributed toward our understanding of TGF␤-mediated growth inhibition and ECM deposition, the mechanisms by which TGF␤ controls gene expression remain largely unknown.Numerous studies have characterized the differential expression of specific genes in response to TGF␤, revealing a common link in the ability of TGF␤ to regulate many of these genes through the functions of the AP-1 family of transcription factors. This protein family, which includes the Fos and Jun proteins, binds a specific DNA sequence and facilitates transcriptional regulation (4). The ability of TGF␤ to induce the expression of several genes, including PAI-1, clusterin, monocyte chemoattractant protein-1 (JE͞MCP-1), type I collagen, and TGF␤1 itself depends on specific AP-1 DNA-binding sites in the promoter regions of these genes (3, 5-