TGF-β Signaling and the Renal Tubular Epithelial Cell
Authors: Jeffrey B. Kopp
Journal: Journal of the American Society of Nephrology (2010)

TGF-β is the paradigmatic profibrotic cytokine and has also been implicated in epithelial-to-mesenchymal transition (EMT).1,2 TGF-β signaling involves ligand binding to the serine-protein kinase TGF-β receptor II (TβRII), which then recruits another serine-protein kinase, TbRI, and thereby initiates a signaling cascade. In this issue of the JASN, Gewin et al.3 describe seemingly paradoxic findings: Whereas deletion of TβRII in the murine collecting duct is associated with normal renal development, in ureteral obstruction, there was enhanced rather than the expected diminution of medullary fibrosis. In ureteral obstruction, TβRII null mice manifest more tubular cell flattening (perhaps evidence for EMT), although this was not directly shown, together with more tubular cell loss, more proteinaceous casts, and more interstitial fibrosis associated with collagen I deposition compared with wild-type mice. The authors' search for the responsible mechanisms excluded increased tubular cell apoptosis and increased numbers of inflammatory cells. They demonstrated increased active TGF-β in kidney tissue and used cultured collecting duct cells with deletion of TβRII to demonstrate that increased activation of integrin αvβ6 was responsible for the observed increase in TGF-β activity. Furthermore, they presented evidence that RhoA activates in TβRII null cells, which is relevant because Rho activation associates with activation of integrin αvβ6; suppression of RhoA activity leads to less TGF-β. Gewin et al.3 suggest a possible mechanism involving Par6. TbRI is localized to tight junctions in polarized epithelial cells, and the binding of TGF-β to TβRII causes TβRII to localize to the TβRI-Par6 complex. Active TβRII causes Par6 phosphorylation and RhoA degradation in epithelial cells4; thus, the absence of constitutive TβRII signaling could account for chronic Rho activation and, in response to injury, could enhance tubular epithelial cell damage with fibrosis. How do we reconcile the seemingly contradictory findings that, on the one hand, TGF-β signaling promotes epithelial injury (including EMT and fibrosis) and, on the other hand, TGF-β signaling is required to sustain a normal epithelial phenotype or at least limit injury in the face of insult? One possibility is that the dosage makes the poison, and, in this case, the dosage must be just right, not too low and not too high. TGF-β has a widely known role in promoting fibrosis.5 Supraphysiologic tissue levels of TGF-β act directly and by mediators such as connective tissue growth factor to increase expression of extracellular matrix proteins, decrease expression of matrix-degrading enzymes, and increase expression of inhibitors of matrix-degrading enzymes. Supraphysiologic levels of TGF-β also have a widely known role in promoting epithelial injury, including EMT. Interestingly, TGF-β also has an essential role in maintaining epithelial cell homeostasis. The homeostatic challenge model, proposed by Wakefield and Stuelten,6 was developed from experimental data obtained from stratified epithelia, such as those found in skin, the gastrointestinal tract, and the ureters and bladder, and proposes distinct activities for TGF-β in different contexts. Under baseline conditions, TGF-β has a custodial function by which its antiproliferative action antagonizes the proliferative effects of endogenous mitogens. When the epithelium is challenged by mechanical injury or inflammation and the level of endogenous mitogens increases, TGF-β plays a first-responder role, selectively increasing apoptosis to counteract cellular proliferation. By fine-tuning epithelial hyperplasia, TGF-β helps to maintain epithelial structure and barrier functions. This model puts in context the findings of Guasch et al.,7 who found that mice lacking TβRII in certain stratified and glandular epithelia rapidly develop spontaneous squamous carcinomas but only in the anogenital region. Wakefield and Stuelten6 proposed that under baseline conditions, the loss of TβRII-mediated signaling associates with a latent migratory state. In mice with selective deletion of TβRII, chronic trauma, which is typical for the anogenital region in mice, results in increased proliferation (unopposed by TGF-β–mediated apoptosis) and the unmasking of the migratory and invasive phenotype (perhaps by Rho activation, as discussed already), leading to malignancy. The relevance of the homeostasis challenge model in understanding the role of TGF-β signaling in the collecting duct epithelium is unclear but is congruent with evidence that physiologic TGF-β signaling acts constitutively by Par6 limiting Rho activation to promote epithelial homeostasis and that supraphysiologic TGF-β signaling acts by activation of Rho family members to induce epithelial injury, including EMT.8,9 Par family proteins are critical for inducing and maintaining epithelial polarity.10 In epithelial cells, the Par6 protein localizes to tight junctions. TGF-β signaling by TβRII increases Par6 phosphorylation and binding, which in turn promotes the localization of the ubiquitinylation protein Smurf1 to the polarity receptor complex and promotes Smurf1 activation. Active Smurf1 facilitates degradation of Rho. By contrast, Rho family proteins, particularly when their activity is increased over the basal state, promote loss of epithelial integrity and EMT. In MDCK cells grown under three-dimensional culture conditions to form polarized cysts, activation of RhoA by blockade of β1 integrin or expression of dominant-negative Rac increases Rho kinase 1 activity and nonmuscle myosin IIA activity and thereby brings about reversal of epithelial polarity.11 In cultured human proximal tubular epithelial cells, reduction in basal activity levels of RhoA and RhoC reduces transcription and stability, respectively, of E-cadherin, whereas in the setting of EMT induced by TGF-β, increased activity of RhoA associates with the expression of α-smooth muscle actin, and RhoC associates with increased stress fibers and cell migration.12 Thus, there is a balance of forces regulating Rho isoform activities, an important determinant of tubular epithelial cell orientation and function. These forces include basal levels of TGF-β signaling maintaining Par6 activity and higher levels of TGF-β signaling promoting Rho activity. In conclusion, previous evidence suggests that pathologic levels of TGF-β signaling trigger tubular cell injury and fibrosis. Gewin et al.3 have demonstrated that a deficiency of TGF-β signaling may have similar effects; thus, a precisely regulated level of TGF-β signaling seems necessary for epithelial cell homeostasis and optimal structure and function. As the authors suggest, these findings may have implications for the development of therapeutic antagonists of TGF-β activity, including antibodies and small molecules, in that the level of inhibition must be precisely calibrated to maintain cell function while inhibiting cell injury and fibrosis. Disclosures None. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.