EMT of renal tubular epithelial cells has been described in both animal models and TGF-β1–treated cells in culture (7
). Here we demonstrate that Smad3, a signaling intermediate downstream of TGF-β and activin receptors, is essential both for TGF-β1–induced EMT of cultured renal tubular epithelial cells and for EMT following UUO in vivo. In both of these systems, expression of all markers of EMT is blocked in the absence of Smad3, thus blocking the formation of fibrogenic myofibroblasts from epithelial precursors.
Experiments utilizing mutated forms of the TGF-β type I receptor unable to bind and activate Smad proteins have clearly shown that the Smad pathway is necessary but not sufficient for induction of EMT by TGF-β (22
), and that other pathways involving phosphatidylinositol 3-kinase, Rho-A and p38MAPK pathways are probably also required (24
). Although these experiments do not differentiate between Smad2 and Smad3, we have recently shown that EMT of lens epithelial cells in response to injury in vivo is completely blocked in the absence of Smad3 (S. Saika, et al., manuscript submitted for publication). Interestingly, in this regard the TGF-β–dependent EMT of cardiac endothelial cells required for the formation of endocardial cushions in the atrioventricular canal of the developing heart is not affected in the Smad3-null mice. This EMT requires expression of the type III TGF-β receptor and may utilize signaling pathways different from those involved in mediating injury-induced EMT (27
Although Smad2 and Smad3 are each activated by the TGF-β and activin receptors, they have very different effects on gene transcription (29
). Somewhat surprisingly, studies in mouse embryo fibroblasts showed that deletion of Smad3 did not affect endogenous levels of Smad2 or its phosphorylation, and vice versa (29
). Thus, EMT of renal epithelial cells following UUO is probably independent of Smad2. Smad3 is critical in mediating the effects of TGF-β on the elaboration of extracellular matrix components, including the synthesis of collagens by fibroblasts (30
), and its loss affords protection from radiation-induced fibrosis (31
) and bleomycin-induced pulmonary fibrosis (32
), presumably by interrupting the pathways necessary for matrix production by fibroblasts. Here we show that Smad3 plays an even more essential role in fibrosis initiated by EMT, as it is also required for generation of the fibrogenic myofibroblasts from epithelial precursors.
The Snail family of zinc-finger transcription factors are strong repressors of transcription of the E-cadherin
gene and are implicated in both physiological and pathological EMT (18
). Recent studies in mouse embryo fibroblasts have identified Snail as an immediate-early gene target of the TGF-β1/Smad3 pathway (E.P. Bottinger, personal communication). Our data, which show that expression of Snail is blocked by neutralizing antibodies against TGF-β in cultured cells in vitro and that EMT is blocked in any condition that interferes with expression of Snail, including loss of Smad3, suggest that it is a critical early-response gene in the TGF-β–driven EMT resulting from UUO.
All the data obtained after UUO in vivo and from mechanical stress–induced EMT of renal tubular epithelial cells in vitro suggest that EMT is initiated by TGF-β1 produced by the renal tubular cells. Especially convincing are the data showing that a neutralizing antibody against TGF-β1 blocked both the increase in TGF-β1 mRNA and the subsequent EMT of mechanically stressed renal epithelial cells in culture, demonstrating that TGF-β1, and not mechanical force per se, initiates the EMT of the stretched cells. Moreover, the absence of TGF-β1 induction in Smad3-null mice implicates this pathway in injury-induced elaboration of TGF-β1 and amplification through a positive-feedback autoinductive loop, similar to that previously reported in monocytes and fibroblasts (29
). This process may be initiated by activation by mechanical stress of latent forms of TGF-β1 secreted constitutively from renal tubular epithelial cells and sequestered by the matrix. In support of this, endothelial and vascular smooth muscle cells reportedly secrete a higher amount of tissue plasminogen activator (t-PA) in response to shear stress (36
). Thus renal tubular epithelial cells facing high-pressure backflow of urine by UUO may also facilitate activation of latent TGF-β1 through any of many pathways described, including proteolytic activation by generation of plasmin from plasminogen by t-PA (38
), or nonproteolytic mechanisms involving thrombospondin-1 (40
) or αV
TGF-β is one of the most potent cytokines known for chemotaxis of monocytes (43
). The significantly reduced levels of monocytes in Smad3-null kidneys following UUO implicates both endogenous TGF-β and the Smad3 pathway in the influx of inflammatory cells in this injury model. As Smad3-null monocytes also show impaired autoinduction of TGF-β1, the reduced inflammatory influx probably contributes secondarily to the reduced levels of TGF-β1 following UUO. Exogenous wild-type but not Smad3-null monocytes, either cocultured with renal tubular epithelial cells in vitro or transplanted into the obstructed kidney in the UUO model in vivo, facilitated EMT, providing evidence that the monocyte influx in UUO contributes to the pathogenesis of the developing fibrosis. In summary, the present results demonstrate that selective ablation of the Smad3 signaling pathway blocks EMT of renal tubular epithelial cells and subsequent pathologic accumulation of matrix proteins while presumably preserving other Smad3-independent TGF-β1 signaling arms. This provides a therapeutic rationale for the development of putative small molecular weight inhibitors of Smad3 that may have fewer side effects than either anti-ligand or anti-receptor approaches, which block all downstream signaling (45
). Our data suggest that selective inhibitors of the Smad3 pathway may prove highly effective in a wide range of fibrotic disorders, including not only obstructive nephropathy and chronic interstitial nephritis but also pulmonary and hepatic fibrosis.