Unified Division of Nephrology, Department of Medicine, Albert Einstein College of Medicine and Montefiore Medical Center, Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York.
Correspondence to Dr. Erwin P. Böttinger, Albert Einstein College of Medicine, Jack and Pearl Resnick Research Campus, 1300 Morris Park Ave, Bronx, NY 10461. Phone: 718 430-3158; Fax: 718 430-8963;
ABSTRACT. Since discovery over a decade ago of a role for thecytokine TGF-ß as key mediator of glomerular and tubulointerstitialpathobiology in chronic kidney diseases, studies of TGF-ßsignaling in the kidney have focused on the molecular biologyof fibrogenesis. In recent years, glomerular and tubular epithelialcell apoptosis and cellular transdifferentiation have been proposedas putative primary pathomechanisms that may underlie progressionof renal disease. This review describes evidence in supportof nonlinear models and functional roles of TGF-ß signalingin mediating apoptosis and epithelial-to-mesenchymal transdifferentiation(EMT) in chronic progressive renal disease. Emphasis is placedon cell context-dependent models of TGF-ß signaling providinga conceptual framework to consolidate seemingly distinct pathomechanismsof progression of glomerular and tubulointerstitial disease.E-mail:bottinge@aecom.yu.edu
The progression of chronic renal diseases is an increasinglycommon condition, often leading to complete destruction of functionalkidney tissue and dependency of affected individuals on life-longtreatments with dialysis or renal allograft transplantation(1). Pioneering studies of Border et al. (2–4), Ziyadehand Sharma (5,6), and many others (reviewed in reference 7)indicate a central role for TGF-ß and its downstream signalingcascades in activating cellular pathomechanisms that underliethe progression of renal diseases. In general terms, the TGF-ßsuperfamily consists of secreted peptides, of which the threeTGF-ß isoforms (TGF-ß1, -2, and -3), activins, andbone morphogenetic proteins (BMP) are best known in mammaliandevelopment, homeostasis, and pathobiology. The TGF-ßisoforms are widely expressed and act on virtually every celltype in mammals by engaging a ubiquitous intracellular signalingcascade of SMAD family proteins through ligand-induced activationof heteromeric transmembrane TGF-ß receptor kinases. Receptor-activatedSmad protein complexes accumulate in the nucleus, where theyparticipate directly in transcriptional activation of targetgenes. Figure 1 shows the basic TGF-ß/Smad signaling axis.
Figure 1. A simplified cartoon of the SMAD protein family and the basic TGF-ß/Smad signaling axis. TGF-ß ligand-binding with type II receptor (RII) serine/threonine kinases (constitutively active) induces recruitment of type I receptor (RI) kinases into a heteromeric ligand-receptor complex, leading to activation of RI kinase. The activated RI then signals to SMAD family members by phosphorylation of two COOH-terminal serine residues, which are conserved in the receptor-regulated subgroup of Smads, R-Smads (Smad1, 2, 3, 5, and 8). Smad1, 5, and 8 are substrates of bone morphogenetic proteins (BMP) receptor kinases. Smad2 and 3 are substrates of TGF-ß and activin receptor kinases. Phospho-Smads interact with cytoplasmic common-partner Smad (Co-Smad), Smad4, which lacks COOH-terminal serines and is not phosphorylated. R-Smad/Co-Smad complexes translocate to the nucleus and bind to specific DNA sequence motifs in target genes to participate in transcriptional activation. R-Smads and Co-Smads are characterized by two major domains of homology, MH1 and MH2, which are connected by a variable linker region. A subfamily of inhibitory Smads (I-Smads) lacks MH1 domains and consists of Smad6 and Smad7, which inhibit TGF-ß/Smad signaling.
Although this seemingly simple linear model has emerged in recentyears as a central view of the TGF-ß/Smad signaling axis(8), it has become clear that it is insufficient to explainthe extensive and context-dependent multifunctionality of TGF-ßthat is a hallmark of these peptides (9,10). Thus, TGF-ßare considered important regulators of cell proliferation, differentiation,apoptosis, immune response, and extracellular matrix remodeling,depending on physiological context. In light of this multifunctionality,it is not surprising that an astonishing array of differentcytoplasmic and nuclear proteins and mechanisms have been discoveredduring recent years, which are exerting direct or indirect agonistor antagonist transmodulation on the central TGF-ß/Smadsignaling axis. In addition, it is increasingly apparent thatTGF-ß receptors can activate Smad-independent signalingmechanisms, although it remains unclear how non-Smad pathwaysare connected to TGF-ß receptors at a molecular level(11). A comprehensive overview of the emerging complexity ofTGF-ß/Smad signaling transmodulators is shown in Figure 2,which is extensively annotated in the corresponding figurelegend. Discussion of molecular details of individual TGF-ß/Smadtransmodulator action is clearly beyond the scope and intentof this review. The interested reader is referred to outstandinggeneral reviews, emphasizing molecular details of the emergingcomplexity and context-dependence of TGF-ß/Smad signaling(see references 12–15). A major focus of the present reviewis to evaluate critically the TGF-ß/Smad signaling systemin the context of renal disease progression, not as a monolithic,linear inducer of fibrogenesis, but rather as a segment of dynamicallyregulated, versatile signaling networks.
Figure 2. Mediator and modifier steps regulating the TGF-ß/Smad signaling axis. Green arrows indicate agonist, and red arrows/lines indicate antagonist function of the depicted steps, respectively. Upon activation of latent TGF-ß complexes and release of TGF-ß ligand in the extracellular space, ligand binding induces heteromeric TGF-ß receptor complexes and type I receptor kinase activity. Recruitment and C-terminal phosphorylation of cytoplasmic R-Smads, Smad2 and Smad3, by activated TßRI is regulated by cytoskeletal proteins and multiple proteins with scaffold, adaptor, anchoring, and/or chaperone function (pink boxes). Green lettering and (+) indicate positive regulation. Red lettering and (-) indicate negative regulation of TGF-ß/Smad signaling. Activated R-Smads and cytoplasmic Co-Smad Smad4 form heteromeric complexes that translocate to the nucleus via nuclear transporters (yellow box). Nuclear Smad complexes bind to consensus Smad3/4 DNA binding elements (SBE) (beige box) in TGF-ß/Smad immediate-early target genes. Example SBE core sequences and their respective positions in select TGF-ß target gene promoters are shown (beige box). Smads regulate target gene transcription together with nuclear transcription factors (brown box), co-activators (grey box), and/or co-repressors (olive box). A list of gene symbols for new immediate-early TGF-ß target genes, identified by microarray screens (11), is shown (green box). Gene symbols can be searched in NCBI Unigene (http://www.ncbi.nlm.nih.gov/UniGene/). Gene activation may be limited and/or terminated by targeting of nuclear R-Smads for proteasome degradation via E2 or E3 enzyme complexes (light blue box). Cytokine and TGF-ß–inducible I-Smads inhibit the TGF-ß/Smad signaling axis by competitive interaction with R-Smads and TßRI, and/or by recruitment of Smurf ubiquitin-ligases (light blue boxes) to activated TßR complexes, leading to its removal via proteasomal degradation. Receptor tyrosine kinase–activated MAPK Erk may phosphorylate motifs in the linker regions of R-Smads to prevent nuclear translocation of activated Smad complexes and inhibit the TGF-ß/Smad signaling axis. Extracellular modulators: TSP1, thrombospondin 1; LAP, latency-associated peptide; IFN-, interferon ; TNF-, tumor necrosis factor ; IL-1ß, interleukin 1ß; HGF, hepatocyte growth factor; EGF, epidermal growth factor. Cross-regulating signal transducers: Stat1, signal transducer and activator of transcription 1; NF-B, nuclear factor–B; Erk, extracellular signal-regulated kinase. Chaperones/Anchors/Adaptors: SARA, Smad anchor for receptor activation; Hrs/Hgs, hepatic growth factor-regulated tyrosine kinase substrate; Cav-1, caveolin-1; Dab-2, disabled-2; SNIX, sorting nexin; STRAP, serine-threonine kinase receptor-associated protein; TRAP-1, TGF-ß receptor-associated protein-1. Nuclear transporters: Crm1, chromosome region maintenance 1. Ubiquitin ligases: Ubc3, ubiquitin conjugating enzyme 3; UbcH5b/c, ubiquitin conjugating enzyme H5b/c; ROC1-SCF(Fbw1a): ROC1, Skp1, Cullin1, and Fbw1a complex. Transcription factors: AR, androgen receptor; ATF-2, activating transcription factor-2; BF-1, brain factor-1; E1A, early region 1A; ER, estrogen receptor; Evi-1, ectopic viral integration site-1; FAST/FoxH1, forkhead activin signal transducer; Gli3, glioblastoma gene product 3; GR, glucocorticoid receptor; HNF4, hepatocyte nuclear factor 4; LEF/TCF, lymphoid enhancer factor/T-cell factor; MEF2, myocyte enhancer-binding factor 2; Menin, multiple endocrine neoplasia-type 1 tumor suppressor protein; Miz1, Myc interacting zinc finger protein 1; OAZ, Olf-1/EBF associated zinc-finger ; PEBP2/CBFA/AML, polyoma-virus-enhancer-binding protein/core-binding factor A/acute myeloid leukemia; SNIP1, Smad nuclear interacting protein 1; TFE3, transcription factor mu E3; Sp1, specificity factor 1; Sp3, specificity factor 3; VDR, vitamin D receptor. Co-Activators: MSG1, melanocyte-specific gene 1; P/CAF, p300/CBP-associated factor. Co-Repressors: SnoN, ski-related novel gene; TGIF, TG-interacting factor; HDACs, histone deacetylases; Ski, Sloan-Kettering Institute proto-oncogene.
Reevaluating Paradigms of Renal Disease Progression
Excessive renal fibrogenesis, the process leading to tissuefibrosis, is considered a dominant pathomechanism induced byTGF-ß in the kidney, largely on the basis of the observationthat glomerular and tubulointerstitial scarring are universaloutcomes of renal disease progression. In part, the currentscientific focus on renal fibrogenesis may be attributable tothe availability of molecular targets, i.e., extracellular matrixcomponents, and assays that allow renal researchers to monitorfibrotic reactions easily at a molecular and whole organ level.In contrast, it has been more challenging to examine cellularpathomechanisms, such as apoptosis, proliferation, and transdifferentiation,largely because chronic progressive kidney disease in humansfrequently is a protracted, focal, and/or segmental process,making it difficult to detect significant single cell alterations.However, along with readily apparent renal scarring, a strikingphenotype of chronic progressive kidney disease is the disappearanceof differentiated glomerular, tubular, and vascular cells thatconstitute the normal nephron (16). Thus, tubular cell atrophyand dilatation are invariably associated with progressive nephronloss and onset of tubulointerstitial fibrosis, a robust histopathologicpredictor of chronic progressive renal disease (17,18). In addition,loss of peritubular capillaries is noted in progressive renaldisease in humans (18). Indeed, a chronic hypoxia hypothesishas been proposed, stating that abnormal post-glomerular hypertensionand vasoconstriction reduce peritubular capillary blood flowand cause rarification of peritubular capillaries, resultingin local hypoxia and tubular atrophy (19). Other reports suggestthat tubular atrophy and interstitial fibrosis may be inducedby increased protein content of ultrafiltrate, giving rise toan inflammatory reaction and interstitial fibrosis (20,21).Upregulation of TGF-ß is consistently associated withthese post-glomerular events, irrespective of the proposed modelof pathogenesis (22–24).
Sclerosing glomeruli are characterized by progressive depletionof podocytes (16,25) and striking loss of glomerular capillariesassociated with depletion of endothelial cells (26–28).Kriz et al. (25) proposed that podocyte depletion and/or detachmentlead to subsequent synechiae formation and tuft adhesions asinitial lesions of glomerular injury, consistent with the frequentlyobserved segmental nature of early glomerular disease. Theirconcept of podocyte depletion as initiating lesion in glomerulosclerosisis further supported by observations in humans with diabetes.Podocyte numbers are reduced in otherwise normal appearing glomeruliand predict long-term urinary albumin excretion (29). Together,these phenotypic observations suggest that segmental loss ofdifferentiated podocytes and tubular and microvascular endothelialcells may be initial and irreversible lesions in progressivenephron loss. Thus, we propose that a comprehensive model ofprogression of renal disease must provide pathomechanisms toaccount for progressive loss of differentiated renal cells.
The common fibrocentric paradigm hypothesizes that fibrosisis the primary pathomechanism mediating renal disease progressionand that the primary pathogenetic role of TGF-ß signalinglies in promotion of fibrogenesis (30). However, in the contextof the aforementioned observations, this paradigm is apparentlyinsufficient in that it fails to provide molecular and cellularlinks to explain the loss of differentiated renal cells. Thereis no direct experimental evidence to support the central tenetof the fibrocentric paradigm, namely that expanded and/or alteredextracellular matrix leads to demise of differentiated renalcells and ultimately organ dysfunction. Interestingly, recentanalyses of a classical condition of excessive scarring, systemicsclerosis, suggest that autoantibody-mediated endothelial cellapoptosis is a primary pathomechanism leading to secondary scleroticskin and organ destruction (31,32). Other conditions associatedwith excessive scarring, such as posttraumatic hypertrophicscars and desmoplastic tumors, are characterized by fibroplasiaand increased vascularization, indicating that extracellularmatrix expansion per se is not sufficient to induce atrophyand/or depletion of resident cells (33,34). If renal fibrosisis not causing nephron loss, what is the evidence for alternativepathomechanisms mediated by TGF-ß? Interestingly, in contrastwith renal research, studies of TGF-ß/Smad signaling innon-renal diseases commonly examine cellular responses otherthen fibrogenesis, such as cell cycle control, apoptosis, anddifferentiation. Can nephron loss thus be explained in parton the basis of apoptosis and/or transdifferentiation inducedby TGF-ß signaling?
There is indeed increasing evidence indicating a role for apoptosisin tubular epithelial cells, causing tubular atrophy in variousexperimental models and human forms of progressive renal disease,including chronic obstructive nephropathy (CON), cyclosporine-inducedallograft nephropathy, diabetic kidneys, and others (35–37).In these studies, TGF-ß1 expression was associated withapoptotic tubular cells. Angiotensin II blockade, or inhibitionof TGF-ß using anti-TGF-ß antibodies, reduced tubularepithelial apoptosis and reduced the extent of tubular atrophyin models of CON and diabetic kidneys (38,39). Interestingly,a detailed analysis from our group of tubulointerstitial lesionsin TGF-ß1 transgenic mice, a model of progressive glomerulosclerosisand tubulointerstitial fibrosis (40,41), indicates that increasedtubular cell apoptosis precedes manifestations of tubular atrophy,tubular dilatation, and perivascular inflammation [Wenjun Ju,Markus Bitzer, and Erwin Böttinger; personal communication,August 2002]. Work by Neilson and colleagues and others (42–44)suggests that tubular epithelial cells may transdifferentiateto acquire (myo)fibroblast phenotypes associated with interstitialfibrosis in experimental models and human renal biopsies (45).Because TGF-ß is a well-known inducer of epithelial-to-mesenchymaltransdifferentiation in several organs (11,46,47), it is perhapsnot surprising that recent reports also implicate a direct rolefor TGF-ß in mediating EMT of renal tubular epithelialcells in vitro and in vivo (48,49).
As described before, glomerulosclerosis in animal models andhumans is characterized in part by depletion of visceral epithelialcells (podocytes) (25,50). Mechanical detachment of podocytesfrom glomerular basement membranes (GBM) and loss in urinaryspace, possibly due to altered cell adhesion and/or increasedmechanical stress of injured podocytes, has been proposed asa potential mechanism. In addition, it is thought that podocytesare unable to proliferate and to replace the "lost" podocytesin most forms of glomerular injury, leading to a state of relativepodocyte insufficiency (25,51,52). We have shown that TGF-ßand Smad7 synergize to induce apoptosis in podocytes in vitro(53). Our in vivo studies indicate that time of peak podocyteapoptosis coincides with expression of TGF-ß1 and Smad7,and with the onset of albuminuria, but precedes mesangial expansionin a TGF-ß1 transgenic model of progressive glomerulosclerosis(53). Recent results from our lab, obtained in CD2AP knockoutmice, a new murine model of focal segmental glomerulosclerosis(54), are similar to and confirm these observations in a second,independent experimental model of chronic progressive glomerulosclerosis[Mario Schiffer, Andrey Shaw, and Erwin Böttinger; personalcommunication, August 2002].
Progressive renal disease is characterized in part by a progressiveloss of the glomerular and peritubular microvasculature (27).Loss of glomerular capillaries is associated with increasedapoptosis of glomerular endothelial cells and correlates withthe development of glomerulosclerosis in rodent models of progressiveanti-GBM disease, in remnant kidney models, and in aging kidney(28,55,56). Similarly, endothelial cell (EC) apoptosis may underliethe loss of peritubular capillaries characteristically associatedwith tubulointerstitial fibrosis and tubular atrophy (57). Severalreports demonstrate that TGF-ß and thrombospondin 1 (TSP-1),a putative activator of extracellular TGF-ß, induce apoptosisin microvascular endothelial cells, including glomerular endothelialcells (58,59). In addition, loss of paracrine signaling of angiogenicsurvival factor vascular endothelial growth factor (VEGF), actingon endothelial cells, enhances EC susceptibility to undergoapoptosis and causes EC growth arrest (27). Loss of expressionof VEGF in glomerular diseases and progressive renal diseasehas been described (27). VEGF is constitutively and selectivelyexpressed in podocytes and tubular epithelial cells in normalkidneys (60,61); therefore, TGF–ß-induced depletionof podocytes and/or tubular epithelial cells may be causingthe decreased VEGF expression. TGF-ß may thus indirectlypromote EC apoptosis by depleting VEGF-producing cell typesin progressive renal disease.
Revised Model of TGF-ß-Mediated Pathomechanisms of Progressive Nephron Loss
On the basis of these observations, we propose a substantiallyrevised model, including new functional roles and multiple pathogeneticendpoints, of TGF-ß signaling in renal disease progression(Figure 3). Whereas interstitial and mesangial matrix expansionmay result from direct activation of "fibrogenic" signalingnetworks by TGF-ß in mesangial cells and interstitial(myo)fibroblasts, additional primary pathomechanisms mediatedby TGF-ß signaling should include apoptosis and EMT. Thus,TGF-ß signaling may initiate pro-apoptotic effectors and/orEMT in tubular epithelial cells, resulting in tubular degenerationand tubular atrophy (Figure 3). In addition, TGF-ß–inducedEMT may convert tubular epithelial cells into activated (myo)fibroblasts,which may be responsible for increased deposition of interstitialmatrix in response to TGF-ß/Smad signaling (Figure 3).Decline of tubular VEGF expression as a result of tubular degeneration/atrophymay indirectly contribute to decreased EC survival in peritubularcapillaries and peritubular capillary loss (Figure 3). Thus,nonlinear, response-specific TGF-ß signaling networksmay lead to tubulointerstitial fibrosis by inducing multiplepathogenetic processes in tubular and microvascular cells. Inaddition, we propose that podocyte and endothelial cell apoptosisare directly induced by TGF-ß signaling in glomeruli exposedto various forms of injury (Figure 3). Apoptosis of podocytesmay lead to depletion of podocytes and formation of synechiaebetween bare GBM and Bowman’s capsule. This results insegmental tuft adhesions characteristic of early lesions inprogressive glomerulosclerosis (Figure 3). Concomitant lossof glomerular capillaries may be a result of decreased EC survivalcaused by direct, pro-apoptotic signaling networks of TGF-ßin EC and by a decrease of VEGF associated with progressivepodocyte depletion (Figure 3).
Figure 3. Profile of putative TGF-ß–mediated response-specific cellular pathomechanisms in progressive nephron loss. See text for details.
This model of multiple pathogenetic events mediated by TGF-ßfurther implies engagement of distinct signaling modules/networksfor the TGF-ß/Smad segment to specify pro-apoptotic signalsin podocytes, tubular epithelial cells, and/or endothelial cells,as opposed to EMT signaling cascades in tubular epithelial cellsor pro-fibrotic signals in mesenchymal myofibroblasts and/ormesangial cells. The proposed shift of functional endpointsof TGF-ß/Smad signaling from fibrosis to apoptosis andEMT as putative primary pathomechanisms for progressive nephronloss has important implications for future experimental approachesand selection of therapeutic targets in progression of renaldisease. In the remaining sections, we review recent advancesin our understanding of distinct, response-specific signalingnetworks activated by TGF-ß/Smad to mediate EMT and apoptosis.
TGF-ß Signaling Networks in Epithelial-to-Mesenchymal Transdifferentiation
Activation of TGF-ß signaling is sufficient to induceEMT in cultured epithelial cells, including a non-transformedmouse mammary cell line (NMuMG) and human keratinocytes (HaCat)(11,46,62,63). A role for EMT in tubular atrophy and appearanceof myofibroblasts in renal disease was first proposed severalyears ago (64). However, evidence for TGF-ß as mediatorof renal tubular EMT has only recently been reported (49,65).For example, advanced glycation end products (AGE) were foundto induce EMT in vitro and in diabetic rats through activationof TGF-ß signaling, indicating an important role for thisTGF-ß–induced response in progression of diabeticnephropathy (49). On the basis of recent studies of signalingpathways activated by TGF-ß to induce EMT in various typesof epithelial cells, a model of this response-specific TGF-ßsignaling network is emerging (Figure 4). EMT is a coordinatedcellular response that involves several distinct processes,including disruption and disassembly of desmosomes and E-cadherinadherens junctions, remodeling of actin cytoskeleton and stressfiber formation, alteration of cell-matrix adhesion, and increasein cell motility (Figure 4). To date, Smads have been implicatedin some aspects of TGF-ß–induced EMT. For example,overexpression of Smad2, Smad3, and Smad4 in NMuMG murine mammarygland epithelial cells induces stress fiber formation (46).In addition, TGF-ß induces Net1A, a RhoA-specific guanineexchange factor, in a Smad-dependent manner (11,66). Net1A isrequired for TGF-ß–induced F-actin remodeling (66).This is consistent with observations that TGF-ß can rapidlyinduce the activation of the small GTPase RhoA, a regulatorof actin cytoskeleton and adhesion junctions in NMuMG cells(67). Inhibitors of Rho kinase can block F-actin remodelingand relocalization of E-cadherin in adherens junctions inducedby TGF-ß (67). RhoA and phosphatidylinositol 3-kinase(PI3K), signaling through the serine-threonine kinase p160ROCKand Akt/PKB, are both required for TGF-ß–induceddisassembly of cell-cell junctions and F-actin remodeling (47,67).TGF-ß induces Smad-interacting zinc finger protein SIP1(68) and SLUG (11), transcriptional repressors of E-cadherincapable of mediating disassembly of cell adherens junctionsin tubular epithelial MDCK cells. We reported a microarray-basedscreen of transcriptional profiles of TGF-ß–inducedEMT in human HaCat keratinocytes (11). These studies suggesta rapid (within 4 h) and dynamic change in expression of approximately4000 transcripts, including factors with roles in mesenchymalprogenitor cell types, and Wnt and Notch signaling. Our recentresults demonstrate that functional inactivation of -secretase,an activator of Notch receptor, completely prevents EMT inducedby TGF-ß, indicating a role for Notch signaling componentsdownstream of TGF-ß [Jiri Zavadil, Lukas Cermak, and ErwinBöttinger; personal communication, August 2002]. In contrast,chemical inhibition of mitogen-activated protein kinase Erk(MAPK Erk) blocks selectively TGF-ß–mediated regulationof genes with functional roles in cell-matrix interactions,cell motility, and endocytosis (11). This is consistent withfunctional inhibition of TGF-ß–induced disassemblyof adherens junctions and cell motility in the presence of MEK/ERKinhibitor (11).
Figure 4. Signaling modules and genetic programs underlying epithelial-to-mesenchymal transdifferentiation induced by TGF-. TGF-ß is sufficient to induce characteristic processes of EMT, including desmosome disassembly, cell-matrix adhesion remodeling, E-cadherin adherens junction disassembly, cell motility, F-actin remodeling (stress fiber formation), and activation of transitional progenitor cell factors. TGF-ß receptor activated pathways/mediators implicated in EMT include Smads, MEK/ERK, Net1A, RhoA, PI3K, PKB/Akt, p160ROCK, and hypothetical mediators (+/- Other mediators) (shaded boxes). Genes induced by TGF-ß within 4 h (11) are denoted by Unigene Symbols colored in red (searchable at http://www.ncbi.nlm.nih.gov/UniGene/). Genes and pathways are aligned according to time of peak activation. Solid line arrows indicate pathways, programs and cellular responses suggested in reference 11. Broken line arrows show mediators and dependent responses demonstrated in other reports. Dotted line arrows show putative pathways proposed in reference 11.
Signaling networks underlying EMT in many tissues, includingkidney, is a young field. Most studies of this cellular responseare conducted in the context of embryonic development and tumorinvasiveness and metastasis formation. The described knowledgeof TGF-ß signaling mediating EMT has been obtained largelyin these contexts. However, a detailed understanding of themolecular signaling mechanisms that mediate tubular epithelialcell EMT in nephrons exposed to pathogenic stimuli will likelybe essential for development of therapies that can prevent renaldisease progression.
It has been difficult to establish apoptosis as a candidatepathomechanism mediated by TGF-ß in progression of renaldisease, because detection and reliable quantitation of apoptoticcells in chronic progressive renal injury is complicated byseveral factors. First, the half-life of apoptotic cells isonly a few hours, limiting the window of detection. In contrastwith extracellular matrix components, cellular remnants of apoptosisare not accumulating in tissue but are often removed by phagocytosis.In addition, detached apoptotic podocytes and tubular epithelialcells may be flushed away by urine. As a consequence, a lowpercentage of apoptotic cells detectable in a tissue sectionmay be associated with a significant loss of cell mass (69,70).Interestingly, glomerular cell apoptosis is markedly increasedin renal biopsy specimen from patients with IgA nephropathy,with the greatest number, up to one apoptotic cell per crosssection, found in lesions described as "predominantly sclerosing"(71,72). The number of apoptotic cells per glomerular cross-sectioncorrelates with semiquantitative "sclerosis" scores in IgA nephropathyand lupus nephritis (73,74).
TGF-ß can induce apoptosis in many cell types, includingrenal cells (reviewed in references 53,75,76). A review of apoptoticsignaling networks of TGF-ß is best guided by organizationof apoptotic signaling in three phases (Figure 5). An Initiationphase is triggered by stress signals and/or specific factors(including TGF-ß) acting through a subset of receptors.During Initiation and Integration phase, cells balance signalsfrom several signaling pathways (pro- and anti-apoptotic) todetermine whether the Execution phase of cell death should beactivated. Critical regulators of the execution phase are theBcl2 family of proteins, which include anti-apoptotic (e.g.,Bcl-2, Bcl-XL) and pro-apoptotic (e.g., Bax, Bad, and Bid) members.Relative expression levels of Bcl proteins can trigger the irreversibleexecution phase by regulating release of cytochrome c from theouter mitochondrial membrane, leading to activation of effectorprotease cascades of the caspase family.
Figure 5. Signaling networks mediating TGF-ß–induced apoptosis. Apoptosis signaling is comprised of three phases: Initiation, Integration, and Execution. TGF-ß receptor activation (RI/RII) leads to activation of Smad3. Smad3 is required for upregulation of pro-apoptotic signaling mediators death-associated protein kinase (DAPK), TGF-ß–induced early gene (TIEG), TGF-ß–stimulated clone 22 (TSC22), and Smad7. Smad7 inhibits anti-apoptotic signaling by NF-B, and may be responsive to TGF-ß-independent signals from pro-apoptotic factors. Smad7 also activates pro-apoptotic JNK. TGF-ß–induced activation of pro-apoptotic MAPK p38 and JNK may be mediated by MAPK cascades via hematopoietic progenitor kinase 1 (HPK1), TGF-ß–activated kinase 1 (TAK1), and TAK1 activator (TAB1). Daxx interacts with RII and activates JNK. TGF-ß induces apoptosis in murine podocytes through activation of p38 and induction of pro-apoptotic protein Bad, leading to activation of caspases. TGF-ß stimulates nuclear translocation of pro-apoptotic ARTS from mitochondria.
There is growing evidence that Smad3 is an important signalinganchor for apoptotic networks. Thus, TGF-ß–inducedapoptosis is enhanced by overexpression of R-Smads in some cells(77). In addition, a number of target genes of Smad3 appearto mediate TGF-ß–induced apoptosis. Smad3 is requiredfor transcriptional activation of death-associated protein kinase(DAPK), which is essential for TGF-ß–induced apoptosisin human hepatoma cells and Burkitts Lymphoma cells (78). Wealso found Smad3-dependent regulation of DAPK in murine podocytes(Markus Bitzer and Erwin Böttinger; personal communication).In addition, activation of several target genes of TGF-ß,including TGF-ß–induced early gene (TIEG) in minklung epithelial and Hep3B cells, and TGF-ß–stimulatedclone 22 (TSC-22) in gastric carcinoma cells (79–81) havebeen implicated in apoptosis. Because TGF-ß activatestranscription of these genes, it is likely that Smad3 is requiredfor these signals. In addition, TGF-ß–induced activationof pro-apoptotic Bcl member Bad in FaO rat hepatoma cells ismediated by Smad3 (82). Daxx is a Fas-receptor–associatedprotein that can interact directly with TGF-ß type IIreceptor. Daxx activates the JNK pathway and mediates apoptosisinduced by Fas, but dominant negative mutant Daxx or functionalinactivation with Daxx antisense oligonucleotides also inhibitTGF-ß–induced apoptosis in B cells and mouse hepatocytes(83). Thus, Daxx may mediate a direct biochemical connectionbetween the TGF-ß receptors and the apoptotic signalingnetwork.
Several reports implicate MAPK pathways in apoptotic signalingby TGF-ß. Activation of TGF-ß–activated kinase-1(TAK-1), a protein of the MAP kinase kinase kinase family, activatesp38 and JNK signaling in TGF-ß family–induced apoptosis(84). In addition, the upstream activator of TAK1, TAB1, maylink pro-apoptotic MAPK to TGF-ß receptors through receptor-mediatedactivation of hematopoietic progenitor kinase 1 (HPK1) (85,86).p38 signaling is required for TGF-ß–induced apoptosisin murine podocytes (53). It is unclear how TGF-ß receptorssignal to MAPK pathways or HPK1 to induce apoptosis. There isalso evidence that Smad7 may induce apoptosis by activationof JNK (87). Indeed, the I-Smad7 has been implicated in apoptosissignaling by TGF-ß and TGF-ß–independent pathways.TGF-ß–stimulated expression of Smad7 is requiredfor induction of apoptosis via p38 activation in prostatic carcinomacells (88), indicating that Smad7 may function downstream ofTGF-ß in apoptosis signaling networks. However, ectopicexpression of Smad7 can induce apoptosis independently of TGF-ßpathways in murine podocytes (53) and distal tubular epithelialMDCK cells (89). Both reports indicate that Smad7 inhibits nucleartranslocation and transcriptional activator function of NF-B,a major activator of transcription of genes with anti-apoptoticfunctions (53,89). Because Smad7 is regulated by TGF-ßin a Smad3-dependent manner (90), and by other extracellularstress and receptor signals (91–93), it may have an importantrole in enhancing TGF-ß–dependent pro-apoptoticsignaling, as well as inhibition of anti-apoptotic pathwaysindependent of TGF-ß (93). A recent report by Larischet al. (94) identifies a septin family protein, apoptosis-relatedprotein in the TGF-ß signaling pathway (ARTS), which isrequired for TGF-ß–induced apoptosis in rat prostatecells. ARTS is normally localized to mitochondria and translocatesto the nucleus when TGF-ß induces apoptosis. Together,these TGF-ß–regulated signaling mediators of apoptosisparticipate in the Integration and Execution phases of apoptoticsignaling networks by stimulating expression of pro-apoptoticmembers and by suppressing expression of anti-apoptotic membersof the Bcl protein family, respectively (95–97).
Concluding Remarks
In this review, we attempt to provide a rationale for a revisedmodel of TGF-ß signaling in progressive renal disease.The role of TGF-ß in renal fibrosis is widely acceptedand targets of fibrogenic signaling have been discussed (98,99);we have therefore focused on TGF-ß–mediated apoptosisand EMT. Apoptosis and EMT are considered primary cellular pathomechanismsmediated by response-specific TGF-ß signaling networksin various forms of tissue injury. We propose that these mechanismsmay cause progressive loss of differentiated renal cells, ahallmark of chronic progression of renal disease. TGF-ß–inducedapoptosis is likely to have a pathogenetic role in podocytedepletion and glomerulosclerosis, tubular degeneration/atrophy,and loss of glomerular and peritubular capillaries. In addition,EMT induced by TGF-ß may contribute to tubular atrophyand generation of interstitial myofibroblasts, leading to concomitanttubulointerstitial fibrosis. The revised model of TGF-ßsignaling in renal disease is intended to stimulate new perspectiveson selection of scientific and therapeutic approaches to understandand prevent chronic progression of renal disease.
Acknowledgments
We thank members and technicians of our laboratory for stimulatingdiscussions and support. This work was supported with fundingfrom NIH grants RO1 DK56607 and RO1 DK60043 to Erwin P. Böttinger.Markus Bitzer is recipient of a research fellowship from theNational Kidney Foundation of New York/New Jersey.
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Abstract
Introduction
2, and -
-secretase, an activator of Notch receptor, completely prevents EMT induced by TGF-ß, indicating a role for Notch signaling components downstream of TGF-ß [Jiri Zavadil, Lukas Cermak, and Erwin Böttinger; personal communication, August 2002]. In contrast, chemical inhibition of mitogen-activated protein kinase Erk (MAPK Erk) blocks selectively TGF-ß–mediated regulation of genes with functional roles in cell-matrix interactions, cell motility, and endocytosis (11). This is consistent with functional inhibition of TGF-ß–induced disassembly of adherens junctions and cell motility in the presence of MEK/ERK inhibitor (11). 
B, a major activator of transcription of genes with anti-apoptotic functions (53,89). Because Smad7 is regulated by TGF-ß in a Smad3-dependent manner (90), and by other extracellular stress and receptor signals (91–93), it may have an important role in enhancing TGF-ß–dependent pro-apoptotic signaling, as well as inhibition of anti-apoptotic pathways independent of TGF-ß (93). A recent report by Larisch et al. (94) identifies a septin family protein, apoptosis-related protein in the TGF-ß signaling pathway (ARTS), which is required for TGF-ß–induced apoptosis in rat prostate cells. ARTS is normally localized to mitochondria and translocates to the nucleus when TGF-ß induces apoptosis. Together, these TGF-ß–regulated signaling mediators of apoptosis participate in the Integration and Execution phases of apoptotic signaling networks by stimulating expression of pro-apoptotic members and by suppressing expression of anti-apoptotic members of the Bcl protein family, respectively (95–97). 
, tumor necrosis factor 
