Inhibitory Smads and TGF- Signaling in Glomerular Cells

Smad6 and Smad7 are inhibitory SMADs with putative functional roles at the intersection of major intracellular signaling networks, including TGF, receptor tyrosine kinase (RTK), JAK/STAT, and NFB pathways. This study reports differential functional roles and regulation of Smad6 and Smad7 in TGFsignaling in renal cells, in murine models of renal disease and in human glomerular diseases. Smad7 is upregulated in podocytes in all examined glomerular diseases (focal segmental glomerulosclerosis [FSGS], minimal-change disease [MCD], membranous nephropathy [MNP], lupus nephritis [LN], and diabetic nephropathy [DN]) with a statistically significant upregulation in “classical” podocyte-diseases such as FSGS and MCD. TGFinduces Smad7 synthesis in cultured podocytes and Smad6 synthesis in cultured mesangial cells. Although Smad7 expression inhibited both Smad2and Smad3-mediated TGFsignaling in podocytes, it inhibited only Smad3 but not Smad2 signaling in mesangial cells. In contrast, Smad6 had no effect on TGF/Smad signaling in podocytes and enhanced Smad3 signaling in mesangial cells. These data suggest that Smad7 is activated in injured podocytes in vitro and in human glomerular disease and participates in negative control of TGF/Smad signaling in addition to its pro-apoptotic activity, whereas Smad6 has no role in TGFresponse and injury in podocytes. In contrast, Smad6 is upregulated in the mesangium in human glomerular diseases and may be involved in functions independent of TGF/Smad signaling. These data indicate an important role for Smad6 and Smad7 in glomerular cells in vivo that could be important for the cell homeostasis in physiologic and pathologic conditions. The TGFsignaling system controls an array of cellular responses to various forms of chronic glomerular injury, including extracellular matrix accumulation, proliferation, hypertrophy, and podocyte apoptosis (1-5). Increased expression of TGFisoforms and components of the intracellular signaling system is described in most models of glomerular disease and progressive human disease, irrespective of the underlying etiology (6). Together, these studies implicate the TGFpathway as the key molecular signaling cascade involved in mediating cellular pathomechanisms resulting in glomerulosclerosis and interstitial fibrosis. The SMAD family of intracellular proteins consists of several functionally and structurally defined subgroups (reviewed in references 7 and 8). Receptor-activated Smads (R-Smads) are characterized by conserved N-terminal and C-terminal major homology domains, MH1 and MH2, respectively. In this subfamily, Smad1, Smad5, Smad8 act as positive signal transducers activated by interaction with ligand-activated bone morphogenetic protein (BMP) receptors, whereas Smad2 and Smad3 act in the TGF/ activin receptor pathway. R-Smad activation depends on their C-terminal serine phosphorylation by cognate receptor kinases, followed by complex formation with another type of Smad, common partner Smad4, and nuclear translocation and transcriptional activation of target genes. Smad6 and Smad7 lack N-terminal MH1 domain and are the only members of the SMAD subfamily of inhibitory Smads (I-Smads). I-Smads are induced at a transcriptional level by TGFand BMP and may inhibit intracellular signaling emanating from ligand-activated BMP receptors and/or TGFreceptors, providing a mechanism for negative feedback regulation (9). At physiologic concentrations, Smad6 may selectively inhibit BMP receptor signaling, whereas Smad7 inhibits TGF/Activin receptor signaling. Smad7 may function as a general negative regulator of TGFreceptor signaling, inasmuch as Smad7 mediates both autoinhibitory feedback and downregulation of TGFsignaling by major opposing pathways, including Jak1/Stat1 (10) and the NFB pathway (11). Although I-Smads may inhibit R-Smads by forming stable interactions with activated type I receptors, thus preventing phosphorylation/activation of R-Smads by receptor kinase (9,12), recent reports define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGFreceptor for degradation (13,14). Additional activities of I-Smads may include transcriptional repressor function (Smad6) (15) and pro-apoptotic functions (1,16-18). Here we report differential functional roles and regulation of Smad6 and Smad7 in TGFsignaling in renal cells, in murine models of renal disease, and in human Received June 10, 2002. Accepted July 25, 2002. Correspondence to Dr. Erwin P. Böttinger, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. Phone: 718-430-3158; Fax: 718-430-8963; E-mail: [email protected]