Targeted Disruption of Smad3 Reveals an Essential Role in Transforming Growth Factor β-Mediated Signal Transduction

The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor β (TGF-β) 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-β-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-β’s antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-β-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-β to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-β-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-β and implicate Smad3 as a potential effector for TGF-β in modulating immune system function.

Smads蛋白家族由9种同源相关蛋白组成，作为转化生长因子β（transforming growth factor β, TGF-β）配体超家族的信号转导中间分子发挥功能。为阐明该家族成员Smad3的体内生物学功能，我们构建了该基因靶向敲除的小鼠模型。Smad3纯合缺失小鼠虽较野生型同窝仔鼠体型偏小，但可正常存活并发育至成年，且表现出前肢畸形的早期表型。为研究Smad3的细胞生物学功能，我们构建了Smad3纯合缺失小鼠的胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）与皮肤成纤维细胞。实验证实，纯合缺失的MEFs无法形成含Smad的DNA结合复合物，亦无法诱导TGF-β应答启动子载体p3TP-lux的转录。利用原代皮肤成纤维细胞，我们还证实Smad3对于内源性纤溶酶原激活物抑制剂1（plasminogen activator inhibitor 1）的诱导不可或缺。后续研究发现，Smad3纯合缺失的MEFs对TGF-β的抗增殖效应存在部分抗性，由此明确了Smad3在TGF-β介导的生长抑制过程中的核心作用。随后我们检测了Smad3高表达的细胞类型，尤其是免疫来源的细胞。尽管未在Smad3纯合缺失小鼠的免疫系统中发现明确的发育缺陷，但观察到功能性缺陷：TGF-β抑制经特异性刺激激活的脾细胞增殖的能力受损。此外，原代脾细胞在TGF-β介导的细胞因子产生抑制过程中存在功能缺陷。综上，本研究数据明确了Smad3在介导TGF-β抗增殖效应中的核心作用，并提示Smad3可能作为TGF-β的效应分子参与调控免疫系统功能。