INVITED REVIEW
TGF-beta signal transduction and mesangial cell fibrogenesis

H. William Schnaper, Tomoko Hayashida, Susan C. Hubchak, and Anne-Christine Poncelet

Division of Kidney Diseases, Department of Pediatrics, The Feinberg School of Medicine of Northwestern University, and Children's Memorial Institute for Education and Research, Chicago, Illinois 60611-3008


    ABSTRACT
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ABSTRACT
INTRODUCTION
TGF-beta IN PROGRESSIVE RENAL...
TGF-beta SIGNAL TRANSDUCTION
TGF-beta -STIMULATED ECM COLLAGEN...
SMAD SIGNALING IN MESANGIAL...
CROSS TALK BETWEEN SMAD...
INTERACTIONS IN TRANSCRIPTIONAL...
EFFECTS OF TGF-beta ON...
IMPLICATIONS OF TGF-beta SIGNALING...
REFERENCES

Transforming growth factor-beta (TGF-beta ) is closely associated with progressive renal fibrosis. Significant progress has been accomplished in determining the cellular signaling pathways that are activated by TGF-beta . This knowledge is being applied to glomerular mesangial cell models of extracellular matrix (ECM) accumulation. A central component of TGF-beta -stimulated mesangial cell fibrogenesis is the TGF-beta family-specific Smad signal transduction pathway. However, while Smads play an important role in collagen accumulation, recent findings indicate that cross talk among a variety of pathways is necessary for maximal stimulation of collagen expression. Further investigation of these multiple interactions will provide insight into possible ways to interrupt cellular mechanisms of glomerular fibrogenesis.

transforming growth factor-beta ; glomerulosclerosis; Smads


    INTRODUCTION
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INTRODUCTION
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TGF-beta SIGNAL TRANSDUCTION
TGF-beta -STIMULATED ECM COLLAGEN...
SMAD SIGNALING IN MESANGIAL...
CROSS TALK BETWEEN SMAD...
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EFFECTS OF TGF-beta ON...
IMPLICATIONS OF TGF-beta SIGNALING...
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GLOMERULOSCLEROSIS IS A PROCESS by which normal, functional glomerular tissue is replaced by accumulated deposits of extracellular matrix (ECM). It represents a common pathway for the loss of functioning glomeruli associated with primary diseases as disparate as chronic glomerulonephritis, obstructive uropathy, and retroviral infection (88, 90). In addition, it represents either the cause or the outcome of many cases of steroid-unresponsive nephrotic syndrome. Idiopathic focal segmental glomerulosclerosis is a leading cause of chronic progressive kidney disease and appears to be increasing in incidence in both children (8, 95) and adults (47).

The pathogenesis of glomerulosclerosis is uncertain. It is likely that all three major cells of the glomerulus participate in the fibrotic process. Recently published genetic data (11, 44, 75) and the finding of podocyte abnormalities in transgenic models of glomerulosclerosis (93) or in patients (94) suggest that the visceral epithelial cell plays a significant role. This assertion is supported by earlier data implicating potential epithelial cell stressors such as glomerular hypertension, hyperfiltration, or hypertrophy in sclerosis (12). Some models implicate the endothelial cell in the sclerotic process (4, 53). Still others suggest a role for the mesangial cell (25). This last possibility is attractive because, in many models of glomerulosclerosis (as well as in idiopathic focal segmental glomerulosclerosis, clinically), ECM accumulation often appears to begin in the mesangium. In addition, filtered macromolecules may be trapped in the mesangium, initiating an inflammatory response that could play a role in stimulating ECM synthesis. A unifying hypothesis can be constructed that includes participation by all of the cellular elements of the glomerulus. Glomerular capillary hypertension, or a genetic or acquired abnormality of podocyte adhesion or structure, permits hyperfiltration of macromolecules. Paracrine signals from the injured podocyte stimulate endothelial cell expression of leukocyte adhesion molecules and impair endothelial cell fibrinolytic activity. Signals from epithelial or endothelial cells to the mesangium, or direct delivery of proinflammatory substances through the glomerular filtrate, initiates a process that culminates in the accumulation of ECM (89). Mesangial expansion infringes on the capillary spaces, decreasing filtration surface area in the glomerular tuft.

One aspect of this unfortunate series of events is mesangial accumulation of ECM. The critical determinant of matrix accumulation is the balance between ECM synthesis and dissolution (19, 87). This net matrix turnover reflects rates of matrix production (affected by transcription and translation) or degradation (determined by synthesis and activity of ECM proteases and their inhibitors) and factors that affect the susceptibility of the ECM proteins to degradation, such as glycosylation (102) or the stability with which these proteins have been incorporated into the matrix. Recently, efforts have been directed toward modeling the cellular events regulating glomerular ECM turnover. A variety of physiological, pharmacological, and molecular approaches has been used to study how various mediators initiate or modify intracellular signaling pathways to cause mesangial cell matrix accumulation. These factors include transforming growth factor (TGF)-beta (9), basic fibroblast growth factor (28), platelet-derived growth factor (28), ANG II (64), connective tissue growth factor (24), and various eicosanoids (52). This review will focus primarily on TGF-beta .


    TGF-beta IN PROGRESSIVE RENAL DISEASE
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INTRODUCTION
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TGF-beta SIGNAL TRANSDUCTION
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IMPLICATIONS OF TGF-beta SIGNALING...
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A significant body of literature supports a role for TGF-beta in glomerular ECM accumulation. This growth factor is present in human glomeruli and has been associated with increased mesangial matrix in several glomerular diseases (109), including diabetic nephropathy (105). Intrarenal infusion of antisense oligonucleotides to decrease the expression of TGF-beta decreases sclerosis in experimental nephropathy (3). Conversely, infusion of the TGF-beta gene causes sclerosis in rats (40). Mice transgenic for increased expression of TGF-beta develop renal fibrosis (66). These data suggest that TGF-beta could play a role in glomerular ECM accumulation in human disease.

However, not all studies have detected elevated levels of TGF-beta in the glomerulus in human disease or animal models of glomerulosclerosis. Sclerosis represents the final outcome of a number of initiating events that could affect the balance in ECM turnover. In addition to disease heterogeneity, varied data also could reflect the duration of illness. Thus patients studied late in disease progression could be at a stage where a TGF-beta -mediated process has been supplanted by one mediated by another factor such as connective tissue growth factor. Nonetheless, the evidence is strong, albeit circumstantial, that TGF-beta plays a significant role in many cases of glomerulosclerosis.


    TGF-beta SIGNAL TRANSDUCTION
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INTRODUCTION
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TGF-beta SIGNAL TRANSDUCTION
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TGF-beta is a pleiotrophic cytokine that originally was described as permitting anchorage-independent growth in soft agar (a model of neoplastic transformation). It was subsequently found to decrease cell division, suppress certain immune responses, and induce differentiation in some cell types. The mammalian TGF-beta s belong to a larger family of similar molecules that includes activin and the bone morphogenetic proteins (BMPs) (59). The most broadly studied TGF-beta , TGF-beta 1, is secreted as an inactive, 25-kDa homodimer that is noncovalently associated with a latency-associated protein (Fig. 1). In some cases, this complex may be bound to a 125- to 160-kDa latent TGF-beta -binding protein. The latency-associated protein inhibits binding of TGF-beta to its receptor. Dissociation of the potentially active molecule from the complex may be accomplished by a number of environmental triggers, including heat, shear force, pH extremes, and proteolysis (67). The concepts of latency and activation are important and often overlooked in studies of TGF-beta production, secretion, and effects (68). In addition, the local availability of TGF-beta is not usually taken into consideration. It binds to the ECM (perhaps facilitated by the latent TGF-beta -binding protein) with a KD of ~10-8 M, while the KD for the receptor is at least an order of magnitude lower (22). These affinity differences permit the ECM to function as a reservoir, releasing the growth factor when the local concentration falls below the ECM KD while maintaining an ambient level sufficient to bind to the cell receptors. This modulation of local concentration is a critical determinant of the effects of TGF-beta in a given system.


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Fig. 1.   Schematic structure of transforming growth factor (TGF)-beta . The active protein is shown in 2 forms: with the small latent complex including the latency-associated protein (LAP; A) and in the large latent complex associated with the latent TGF-beta -binding protein (LTBP; B).

TGF-beta -family ligands bind to a corresponding family of receptors that is comprised of seven type I receptors (Tbeta R-I) and five type II receptors (Tbeta R-II) (5). Tbeta R-I and Tbeta R-II may form a variety of heterodimers; several of these have been described in renal cells (55). There are some unique aspects of TGF-beta -family signal transduction. First, whereas most growth factor receptors are tyrosine kinases, the TGF-beta -family receptors function as membrane-bound serine/threonine kinases. Second, most receptors signal by activating a cascade of second messengers that transmit signals toward the nucleus, culminating in the activation of a transcription factor; or, in the case of the steroid hormone receptor family, the ligand binds to a cytoplasmic receptor that itself serves as a transcription factor. The TGF-beta -family receptors are unique in that they are integral membrane receptors which bind to and activate proteins that translocate to the nucleus to regulate transcription rather than initiate a cascade. Viewed in terms of function and complexity, these attributes suggest that the mechanism of action of this family of receptors is an evolutionary intermediate between those of the nuclear hormone receptors and the receptor tyrosine kinases.

The proteins that are activated by the TGF-beta -family receptors are called "Smad" proteins. The Smads were originally described simultaneously by the fly and worm scientific communities. In Drosophila melanogaster, a gene product was determined to play a role in embryological patterning regulated by a BMP homologue called decapentaplegic and was termed "MAD" (for "mothers against decapentaplegic") (91). Researchers investigating the developmental genetics of Caenorhabditis elegans identified a series of proteins that, when mutated, produced an identical, small phenotype in the worms (indicating a common effector pathway for these molecules). They termed this family the "Sma" proteins (84). Eventually, a consensus name, Smad, was agreed on.

The Smads have a general structure of two MAD homology (MH) domains connected by a linker region (Fig. 2). The protein-amino acid sequence includes a number of serines and threonines that represent potential phosphoacceptor sites. On ligand binding, Tbeta R-II transphosphorylates Tbeta R-I (Fig. 3). Two receptor heterodimers combine to form a tetramer that binds to a subfamily of Smads termed the R-Smads, which are receptor activated and also pathway restricted (Smad2 and Smad3 for TGF-beta and activin; Smad1, -5, and -8 for the BMPs) (60). Smad2 and Smad3 are recruited to the cell membrane receptor through their affinity for the Smad anchor for receptor activation, a protein that has a binding domain specific for TGF-beta -family receptors (100). The quiescent R-Smads maintain a hairpin configuration until becoming phosphorylated at a COOH-terminal SSXS motif, whereupon they spring open and become active. The activated R-Smads form heteromultimers with a second type of Smad, the common-pathway Smad called Smad4. This complex is translocated to the nucleus, where it participates in transcriptional regulation (76). Initially, it was uncertain whether Smads bound directly to DNA or attached through an intermediary protein complex (14, 113). Subsequently, however, a Smad-binding element, including the sequence CAGA, has been characterized (43, 110). Nonetheless, in most cases transcriptional activation, even when demonstrably Smad dependent, requires the binding of additional transcription factors. Transcriptional cooperation has been demonstrated with proteins such as AP-1 transcription factors (113), Fast-1 (14), Fast-2 (50), TFE3 (35), and Sp1 (18, 51, 112). R-Smads and Smad4 have also been shown to interact with the binding protein for cAMP-response element binding protein/p300 coactivators (92, 104). Inhibiting factors include TRIP-1 (16), SnoN (96), Ski (56), and SNIP (46).


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Fig. 2.   Domain structure of Smad2. * Potential phosphoacceptor sites; MH, MAD homology domain.



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Fig. 3.   Schematic diagram of Smad signal transduction. R-Smads in the cytoplasm (A) are recruited to the TGF-beta receptors (Tbeta Rs; B), where they are phosphorylated (P; C). A complex with the common Smad, Smad4, is formed (D) and translocated to the nucleus, where it regulates gene transcription (E). SARA, Smad anchor for receptor activation.

A third category of Smad proteins, the inhibitory Smads (I-Smads), includes Smad6 (38) and Smad7 (30, 73), which have been identified in the kidney (101). The I-Smads are able to bind to Tbeta R-I but lack critical phosphoacceptor sites and therefore prevent phosphorylation of R-Smads. In addition, Smad6 has been shown to inhibit the binding of Smad4 with R-Smads, decreasing signal in that way as well (29). Smad7 expression is stimulated by TGF-beta in an apparent negative-feedback loop (69).

The definition of this pathway, and of its unique attributes described above, has represented a major advance in our understanding of how TGF-beta -family ligands and their receptors function to transmit signals into the cell. A caveat regarding this progress is that it was accomplished primarily utilizing ectopic molecular expression systems and/or transformed cell lines. Thus much of the initial data focused on the role of Smads in regulating developmentally expressed genes or cell proliferation and differentiation in cancer. The first human Smad mutations were identified in colon (98), pancreatic (26), and lung (70) cancers, and many more have been described. However, transformed cells may not have a full complement of signal-regulating proteins. In addition, when signaling proteins are overexpressed in cells, the requirements for specific activation steps and specific subcellular localization, as well as the potential need for interaction with additional proteins and signaling pathways, may be freed from subtle physiological regulatory constraints (65). Thus it has been important that these pathways should be studied for their function in ligand-activated, nontransformed cells.


    TGF-beta -STIMULATED ECM COLLAGEN PRODUCTION
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Several reports have indicated that TGF-beta stimulates mesangial cell ECM production. In cultured mouse mesangial cells, TGF-beta 1 stimulates production of types I and -IV collagen and fibronectin (57). In rat mesangial cells, TGF-beta 1 has been shown to have variable effects. In one report, it increased proteoglycan synthesis without any changes in collagen and fibronectin synthesis (10, 72), while another group described increased expression of alpha 1(I) collagen and alpha 1(IV) collagen and fibronectin genes (97). TGF-beta 1 also inhibits plasminogen activator production while stimulating plasminogen activator inhibitor synthesis by normal rat glomeruli (99). In various assays examining cultured human mesangial cells or isolated perfused kidneys, TGF-beta 1 stimulates expression of types I, -III, and -IV collagen, laminin, fibronectin, and heparan sulfate proteoglycans (21, 27, 63, 80).

To define more precisely the nature and timing of changes in matrix turnover that are stimulated by TGF-beta , our laboratory evaluated human mesangial cells. TGF-beta 1 increases expression of mRNA for the ECM protease matrix metalloproteinase (MMP)-2, which is paralleled by an increase in its antagonist, tissue inhibitor of metalloproteinases (TIMP)-2. There is no change in TIMP-1 or membrane-type MMP mRNA expression, while the level of MMP-1 mRNA is decreased. At the protein level, TGF-beta 1 increases types I and -IV collagen in both the mesangial cell layer and conditioned media. Many of these changes occur rapidly, with increased expression of collagen mRNAs beginning within 1-4 h of TGF-beta 1 treatment. In contrast to the mRNA studies, MMP-2 and TIMP-1 activity showed little change while MMP-1 did decrease and TIMP-2 was not detected. Together, the net protease activity did not appear to change significantly, suggesting that, for regulating collagen turnover in this short-term cellular model, changes in protease activity are less significant than those in ECM synthesis (80). In other model systems, the regulation of ECM degradation by TGF-beta may be at least as important as ECM synthesis (6).

In addition to the rapid changes in mRNA expression, collagen protein turnover was faster than anticipated. Treatment of near-confluent cells with cycloheximide to inhibit protein synthesis for only 4 h virtually eliminated detectable collagens from the cell layers and conditioned media. While this result could reflect the scorbutic culture conditions (ascorbate, which facilitates the stabilization of collagen fibrils in a matrix, was omitted from the culture medium because it also stimulates collagen synthesis), the results suggested that mesangial cell collagen protein expression in vitro represents a steady-state phenomenon rather than simply ongoing accumulation of synthesized protein. Interestingly, cycloheximide did not affect early TGF-beta 1-induced changes in mRNA expression but reversed increases in alpha 1(I) collagen mRNA at 24 h, suggesting that early changes in expression represent direct effects whereas later changes are mediated by the synthesis of additional proteins. In contrast, alpha 1(IV) collagen mRNA expression did not require intermediate protein synthesis at any time point but was superinduced by cycloheximide treatment. These data indicate that, in human mesangial cells, early changes in net collagen accumulation are regulated primarily at the level of protein synthesis rather than degradation and that the mechanisms of type I collagen and type IV collagen mRNA expression differ significantly (80).


    SMAD SIGNALING IN MESANGIAL CELL ECM ACCUMULATION
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These data suggested that the most appropriate avenue for the investigation of immediate profibrotic cellular events in TGF-beta -stimulated mesangial cells is the examination of ECM synthesis. Toward this goal, we determined whether Smad activity could be linked to mesangial cell collagen expression. Mesangial cells express at least Smad1, -2, -3, -4, and -7. Stimulation of human mesangial cells with TGF-beta 1 induces phosphorylation of Smad2 and Smad3, beginning within 5 min of exposure. Phosphorylation peaks at 30 min but remains present for as long as 24 h (79, 83). The phosphorylation event is paralleled by association among Smad2, -3, and -4. Phosphorylation and association are followed within minutes by increased nuclear localization of this complex. Activity of the TGF-beta /Smad-responsive p3TP-Lux promoter-reporter construct is stimulated by TGF-beta 1 treatment. These data are consistent with the model that ligand-stimulated Smad activation in mesangial cells leads to transcriptional activity in the nucleus, in parallel with previous results derived from the study of transformed cells or overexpressed Smads. The alpha 1(I) collagen and alpha 2(I) collagen promoters are also activated (31, 79); and activation of a collagen promoter-luciferase reporter construct is inhibited by Smad3A, a mutated Smad3 expression construct that lacks critical COOH-terminal serine phosphoacceptor sites and functions as a dominant-negative mutant in this assay (79). Together, these results demonstrate that the Smad pathway is present and functional in mesangial cells and that it can mediate TGF-beta -stimulated collagen I expression. Further support for typical Smad pathway interactions regulating collagen production is provided by the finding of other groups that Smad7 decreases TGF-beta -stimulated mesangial cell collagen production (13, 54). Another way in which Smad7 signaling can affect the sclerosing process is by inducing podocyte apoptosis (85).

It is noteworthy that, while the dominant-negative mutant Smad3A blocks TGF-beta stimulation of COL1A2 promoter-luciferase reporter activity, simply expressing either wild-type Smad3 or Smad3A leads to a large increase (15- and 8-fold, respectively) in basal responses (79). Thus, although the Smad3A construct does not transduce the receptor-activated signal, its overexpression appears to at least partly bypass the requirement for activation. This observation illustrates the concern raised previously about the impact of overexpressing specific proteins on the geometry or the stoichiometry of the cellular signaling response.


    CROSS TALK BETWEEN SMAD SIGNALING AND MAP KINASE PATHWAYS
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Thus the interpretation of these studies can be complicated by technical as well as biological factors. Moreover, investigators examining the response of different cells to an identical stimulus have reported varied and contradictory findings. This outcome has been attributed to myriad differences in experimental conditions, including the culture media used, the question of whether the cells are transformed, the timing and conditions of treatment, etc. However, it is increasingly accepted that such differences may be real and may define tissue specificity in an organism. For example, an endothelial cell will respond differently from a smooth muscle cell, even though the basic signaling pathways in the two cell types are similar. The likely source of this heterogeneity is cross talk among different signal transduction pathways that are present and active to varying degrees in different cells (86). While the paradigm of Smad activation described above is likely to exist in most cells, interaction of Smads with other signals may significantly alter their function. For example, TGF-beta 1 may stimulate a variety of kinases in mesangial cells, including protein kinase A (103) and casein kinase II (111). In rat mesangial cells, TGF-beta has been shown to activate ERK and p38 MAP kinase (15, 36). Activation of p38 has been implicated in TGF-beta 1-stimulated alpha 1(I) collagen mRNA expression (15), whereas ERK has been associated with fibronectin accumulation (39). In human mesangial cell studies from our laboratory, TGF-beta 1 stimulates phosphorylation of both the ERK- and JNK-MAP kinase pathways, but not p38 (31). Biochemical inhibition of ERK reduces TGF-beta 1-stimulated human mesangial cell collagen mRNA expression. Moreover, a dominant-negative ERK construct, but not dominant-negative inhibition of JNK activation, decreases activation of the TGF-beta -specific p3TP-Lux construct as well as a collagen promoter-luciferase reporter construct (31). These results indicate that the ERK-MAP kinase pathway enhances or amplifies Smad-mediated mesangial cell responses.

The interaction of ERK with TGF-beta signaling in mesangial cells is under investigation. Our preliminary data suggest that ERK enhances Smad phosphorylation (32). This finding may contribute further to the controversy in the literature regarding the effect of ERK on Smad activity. Initially, it was felt that ERK activation inhibited Smad signaling (48). More recent studies have supported both the enhancing (20) and inhibitory (49) effects of ERK on Smad activation. Inhibiting ERK decreases Smad-mediated transcriptional activity (31, 32). These differences could reflect the developmental origin of the tissue under study, the nature and intensity of the stimulating and inhibitory signals, the question of whether endogenous or ectopically expressed proteins were studied, or the specific downstream target of Smads that is being evaluated. In renal tubular epithelial cells, TGF-beta stimulates epithelial-to-mesenchymal transdifferentiation (EMT), a pivotal event in some models of fibrogenesis (107) (see below). EMT is inhibited by stimuli of ERK activity (106) or by mediators that have been associated with ERK activation (17). The role of ERK in antagonizing the effects of TGF-beta in renal tubular EMT remains to be fully established.

The non-Smad signaling pathways that are activated after TGF-beta stimulation have been reviewed recently (77). Although they clearly play a role in regulating a number of processes in addition to fibrogenesis (7), the mechanisms by which the TGF-beta receptor mediates these alternative pathways are not well understood. This is an important area for further investigation.


    INTERACTIONS IN TRANSCRIPTIONAL REGULATION
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Another potential site of interaction between Smads and other factors is in the nucleus. As described above, Smad activity likely requires other transcription factors, including an interaction with Sp1 that has been described in several systems (18, 51, 112). In human mesangial cells, TGF-beta 1-induced collagen mRNA expression is inhibited by mithramycin, a blocker of Sp1 DNA binding, but not by curcumin, which inhibits AP-1 transcriptional activity. TGF-beta 1 stimulates interaction of Smad3 and Sp1, and the resulting complex binds to promoter sequences in the COL1A2 gene. Deletion from the promoter of GC boxes that bind to Sp1, or mutation of the CAGA sequence in the Smad-binding element (SBE), abrogates promoter activation by TGF-beta 1. Additional studies showed that the transcriptional activity of the Sp1 transactivation domain B was not induced directly by TGF-beta 1 but instead that this domain became responsive when Smad3 was coexpressed. Thus Smad3 activity is critical to the response, but Sp1 plays a key role in supporting that response (81).

Further insight into these events is provided by elegant studies of laminin-gamma 1 gene regulation by Bomsztyk and colleagues (33, 45). The LAMC1 promoter contains a highly conserved transcriptional element, termed bcn-1. With the use of a yeast one-hybrid approach, the TFE3 transcription factor was cloned from a rat mesangial cell cDNA library, indicating that this basic helix-loop-helix/leucine zipper transcription factor binds to bcn-1. Stimulation of bcn-1 by TGF-beta to activate the LAMC1 promoter was enhanced by overexpression of Smad3 and was dependent on the Smad-binding element in the LAMC1 promoter (45). In addition, a similar strategy identified binding of the gut-enriched Kruppel-like factor (GKLF) to bcn-1. GKLF activity was dependent on synergy with Sp1 (33). Although this second study did not address the role of TGF-beta , the involvement of Sp1 in these events and the responsiveness of GKLFs to TGF-beta -family ligands in other experimental systems (1) suggest that multiple transcription factors, binding to multiple sites, cooperate to induce ECM protein gene expression in response to TGF-beta stimulation. Additional protein-protein interactions at the level of gene transcription include those of Smad with p300, shown in several systems (23, 104), and with estrogen receptor (61).


    EFFECTS OF TGF-beta ON THE ACTIN CYTOSKELETON
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The paradigm of TGF-beta signaling through Smads involves a transcriptional mechanism of action and, by extension, delayed responses. However, other responses may be more immediate in some circumstances, suggesting nontranscriptional actions. For example, TGF-beta 1 rapidly stimulates Ca2+ influx, without promoting Ca2+ release, in SV-40-transformed murine mesangial cells. This response is inhibited by pharmacological inhibitors of inositol-1,4,5-trisphosphate (IP3) receptors and by an antibody to the type III IP3 receptor. Whereas untreated mesangial cells had numerous, spike-like projections on their cell surface, TGF-beta 1 treatment reduced these projections in a Ca2+-dependent manner within 15 min (62).

In preliminary studies, we have found that treatment of human mesangial cells with TGF-beta 1 not only stimulates cytoskeletal rearrangement but also increases incorporation of alpha -smooth muscle actin (alpha -SMA) into stress fibers. Our evidence suggests that these cytoskeletal changes could play a role in collagen expression (Hubchak and Schnaper, unpublished observations). One mechanism for this role could relate to a theory of fibrogenesis that has gained increasing attention: that fibrosis requires resident tissue cells to differentiate into fibroblasts. In atherosclerosis, this has been characterized as vascular smooth muscle cells assuming a "myofibroblastoid phenotype." In mesangial cells, altered collagen expression after TGF-beta treatment has been associated with increased expression of alpha -SMA (37). The phenomenon has been more fully described in the tubular epithelium, where the tubular cell transdifferentiates from an epithelial cell phenotype to a fibroblastoid cell that produces and secretes ECM (107), in a process that also is associated with increased alpha -SMA incorporation into stress fibers (107).


    IMPLICATIONS OF TGF-beta SIGNALING MECHANISMS FOR DISEASE PATHOGENESIS AND TREATMENT
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Taken together, the data presented here suggest that TGF-beta stimulates several pathways in mesangial cells (Fig. 4). In addition to the Smad pathway, Ca2+ influx and MAP kinases are activated; there may be an as yet undetermined hierarchy to these signals. These pathways in turn synergize, perhaps through effects on each other or through parallel tracks into the nucleus, where a variety of transcription factors interact to modulate the transcription of ECM genes. Other events in the glomerulus that may be mediated by Smads, such as endothelial nitric oxide synthase gene expression (108) or cell apoptosis (41, 78, 85), also could affect glomerular function and sclerosis.


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Fig. 4.   Signaling events activated in TGF-beta -stimulated mesangial cell fibrogenesis. Much remains to be determined, such as the mechanism by which the cytoskeleton influences ECM gene expression and the identity of specific inhibitors of ECM gene transcription. IP3, inositol-1,4,5-trisphosphate. For simplicity, the IP3 receptor is shown at the cell membrane, whereas the mechanism by which it increases intracellular calcium is uncertain.

While we have emphasized transcriptional effects of these pathways, posttranslational effects such as alterations in the activity of ECM proteases also could modulate the accumulation of ECM. For example, TGF-beta has been reported to decrease (74) or increase (58) the expression or activity of the MMPs that degrade ECM or the tissue inhibitors of MMPs that slow matrix degradation. Cytochalasin D, a pharmacological agent that disrupts the cytoskeleton, stimulates the activation of MMP-2, increasing collagen degradation (2). Thus the effects of TGF-beta on other biological events that affect ECM turnover, and potential nontranscriptional mechanisms of TGF-beta signaling, are areas of importance that presently are under-studied.

An even more important area of potential investigation is the role of these newly defined pathways in fibrogenesis in vivo. It is clear that increasing TGF-beta expression either locally or systemically (66) causes glomerular or renal fibrosis. Inhibition of TGF-beta binding to its receptor can lessen the degree of experimental renal fibrosis (42). Hong and colleagues (34) have associated the TGF-beta /Smad signaling pathway with an animal model of diabetic renal disease. However, studies of the effects of interrupting Smad signaling on renal fibrogenesis have not been performed. The lack of pharmacological inhibitors of Smad signaling has prevented all but the most rudimentary studies of how Smad signaling might be regulated in vivo. A possible approach might involve new techniques of local gene expression (71, 82) to increase intracellular levels of I-Smads. Significant technical advances will be required for such approaches to be feasible. Thus our expanded understanding of this field over the past several years offers great promise but one that requires considerable additional investment before it will reach fruition.


    NOTE ADDED IN PROOF

Further evidence of a role for inhibitory Smads in glomerular disease is found in an article by Schiffer and colleagues (Schiffer M, Schiffer LE, Gupta A, Shaw AS, Roberts ISD, Mundel P, and Böttinger EP. Inhibitory Smads and TGF-beta signaling in glomerular cells. J Am Soc Nephrol 13: 2657-2666, 2002), reporting altered podocyte I-Smad expression in human disease.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49362 and a grant to Dr. A.-C. Poncelet from the National Kidney Foundation of Illinois.


    FOOTNOTES

Address for correspondence: H. W. Schnaper, Pediatrics W-140, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: schnaper{at}northwestern.edu).

10.1152/ajprenal.00300.2002


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
TGF-beta IN PROGRESSIVE RENAL...
TGF-beta SIGNAL TRANSDUCTION
TGF-beta -STIMULATED ECM COLLAGEN...
SMAD SIGNALING IN MESANGIAL...
CROSS TALK BETWEEN SMAD...
INTERACTIONS IN TRANSCRIPTIONAL...
EFFECTS OF TGF-beta ON...
IMPLICATIONS OF TGF-beta SIGNALING...
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