Transforming growth factor-β1 is a multifunctional cytokine that regulates the growth, differentiation and function of various cell types.84 The TGF-β superfamily includes the various forms TGF-β, bone morphogenic protein, nodals, activin, the anti-Mullerian hormone and many other structurally related factors.85 Also, the TGF-β family includes three factors with similar structures and functions: TGF-β1, -β2 and -β3. TGF-β1 is normally secreted as a latent precursor complex composed of three proteins, including the bioactive peptide of TGF-β1 and latency-associated peptide-β1 (LAP-β1). TGF-β1 forms a complex with LAP-β1 non-covalently, forming the small latent complex (SLC). In this configuration, TGF-β1 is inactive because it cannot bind to its receptors. On the other hand, TGF-β1 activation is a complex process, involving conformational changes induced by either cleavage of the LAP-β1 by various proteases such as plasmin, thrombin, plasma transglutaminase and endoglycosylases, or by physical interactions of the LAP-β1 with other proteins such as integrins and thrombospondin 1, leading to the release of bioactive and mature TGF-β1.6,86–89 Integrin αvβ8 has also been demonstrated to be able to activate latent TGF-β1 by MT1-MMP1-dependent degradation of LAP-β1.90 Taken together, TGF-β activation seems to be largely controlled by its interaction with extracellular or cell surface molecules.
Transforming growth factor-β1 initiates signaling through the ligand-dependent activation of a complex of heterodimeric transmembrane serine and threonine kinases consisting of TGF-β receptor type I (TGF-βRI) and TGF-βRII. TGF-βRII is thought to determine the ligand specificity. TGF-βRI alone is unable to bind TGF-β, and TGF-βRII is unable to signal without TGF-βRI.91 Betaglycan, a transmembrane proteoglycan also known as TGF-βRIII, allows high-affinity binding of TGF-β1 to TGF-βRII, but does not itself transduce signal. Upon binding TGF-β1, the receptors rotate within the complex, resulting in the phosphorylation and activation of TGF-βRI by the constitutively active and autophosphorylated TGF-βRII. TGF-β1 signals from the receptor to the nucleus using a set of Smad proteins (Fig. 2). The activated TGF-βRI directly phosphorylates Smad2 and Smad3. Once activated, Smad2 and Smad3 associate with Smad4 and translocate to the nucleus, where such a complex regulates transcriptional responses of target genes. However, the DNA binding affinity of Smad is not relatively high, and Smad generally require interaction with other DNA binding partners to be stably tethered to DNA.92 Such interactions either influence Smad function directly or, alternatively, affect the functions of its binding partners. Sp1, Ets1, CBP/p300, the AP-1 complex, TFE3, FAST-2, vitamin D nuclear receptor, Gli3 and Evi-1 have all been reported to bind to Smad3 and thereby participate in the regulation of natural or artificial reporter constructs known to be inducible by TGF-β1.53,93 On the other hand, the inhibitory Smad7 associates with ligand-activated TGF-βRI and interferes with the phosphorylation of Smad2 and Smad3 by preventing their interaction with activated TGF-βRI.94 Because the expression of Smad7 is induced by TGF-β1, Smad7 is thought to inhibit TGF-β signaling by a negative feedback system.95 Smad7 also recruits ubiquitin ligases, termed Smurf, to the TGF-β receptor complex to promote its degradation through proteasomal and lysosomal pathways.96 In addition, transcriptional co-repressors, including c-Ski and SnoN also function as another negative feedback system, as described above.
Figure 2. Schematic representation of the autocrine transforming growth factor (TGF)-β signaling cascade in systemic sclerosis (SSc) fibroblasts. The upregulated integrins on the cell surface activate latent TGF-β1 by binding to latency-associated peptide-β1 (LAP). The activated TGF-β1 binds to the overexpressed TGF-β receptor, resulting in constitutive activation of downstream signaling pathways. TGF-β receptor recruits and phosphorylates Smad2 and Smad3, leading to the association with Smad4 and translocation of the formed heterocomplex into the nucleus. The translocated heterocomplex binds to the target gene with other transcription factors, such as Sp1, and regulates gene expression, cooperating with p300/CBP. On the other hand, negative feedback systems mediated by Smad7, Smurf and c-Ski/SnoN are impaired in SSc fibroblasts.
Download figure to PowerPoint
The principal effect of TGF-β1 on mesenchymal cells is its stimulation of ECM deposition. TGF-β1 has been shown to induce myofibroblast differentiation and increase the expression of collagen types I, III, VI, VII and X, fibronectin and proteoglycans.32,87 Accordingly, TGF-β1 has also been implicated as being a key mediator in a number of fibrotic diseases in the skin. In addition, an increased expression for TGF-β1 has been documented during the phase of tissue remodeling in several forms of acute or chronic lung fibrosis.97 TGF-β is also closely associated with renal interstitial fibrosis, cardiac fibrosis and liver fibrosis.
Active and total (active and latent) TGF-β1 levels are significantly elevated in cultured peripheral blood mononuclear cells derived from SSc patients,98 whereas we previously reported that these levels in the culture media of SSc fibroblasts are as high as those of normal fibroblasts.99 However, our following findings may suggest that the activation of dermal fibroblasts in SSc is a result of stimulation by “autocrine TGF-β signaling”, without increasing the concentration of active TGF-β1: (i) although the transcriptional activity of the COL1A2 gene in SSc fibroblasts is constitutive higher than that in normal fibroblasts, the responsiveness to TGF-β1 is decreased in those cells; (ii) the blockade of TGF-β1 signaling with anti-TGF-β antibodies or anti-TGF-β1 antisense oligonucleotides abolishes the increased expression of human COL1A2 mRNA in SSc fibroblasts;99 and (iii) phosphorylated levels and DNA-binding activity of Smad3 is constitutively upregulated in SSc fibroblasts.32,100 Recently, we have reported that upregulated expression of αvβ5 integrin contributes to the establishment of autocrine TGF-β signaling in SSc fibroblasts through activation of endogenous latent TGF-β1. αvβ5 integrin may recruit and activate SLC on the cell surface of SSc fibroblasts (Fig. 2). Recruitment and/or activation of latent TGF-β1 in the pericellular region might enhance the incidence of interaction between active TGF-β1 and its receptors, leading to the self-activation of SSc fibroblasts without increasing amount of TGF-β1.87,101,102
Also, enhanced expression of TGF-β receptors has been well demonstrated in fibrosis.99,103 The overexpression of TGF-β receptors induces collagen transcription in cultured dermal fibroblasts.104 SSc fibroblasts express elevated levels of TGF-β receptors, and this correlates with the elevated levels of COL1A2 mRNA.104 Another study indicates that TGF-βRI : TGF-βRII ratio is increased in SSc fibroblasts, and suggests that it contributes to the aberrant TGF-β signaling in SSc.105 The increased TGF-βRI in SSc fibroblasts might be explained by the increased stability of the receptor by pulse-chase experiment.32 Although there is no significant difference in the expression levels of Smurfs between normal and SSc fibroblasts, the transient overexpression of Smurf or the treatment with exogenous TGF-β1 does not induce significant decrease in the TGF-βRI protein levels in SSc fibroblasts. In addition, simultaneous overexpression of Smurf and the exogenous TGF-β1 stimulation also have no significant effect on the levels of TGF-βRI protein in SSc fibroblasts. Taken together, degradation activity of TGF-β receptors by Smurf may be disturbed in SSc fibroblasts.
Many of the characteristics of SSc fibroblasts resemble those of healthy fibroblasts stimulated by TGF-β1;106,107 for example, increased type I collagen expression, increased phosphorylation of Smad3, or increased interaction of Smad3, Sp1 and p300 as described above. Previously, it was reported that the TGF-β-responsive element of COL1A2 promoter in normal fibroblasts and the sequence involved in the intrinsic upregulation of COL1A2 gene expression in SSc fibroblasts are both located between bp −376 and bp −108 sites.100 This may indicate that the intrinsic upregulation of ECM genes in SSc fibroblasts utilizes the autocrine TGF-β1 signaling-dependent pathway. The autocrine TGF-β signaling may also be the cause of increased Ets1 expression, reduced Fli1 expression, increased c-myb expression, and the increased interaction of p300/CBP with Smad, Sp1 and Ets1 in SSc fibroblasts, as described above.30,34,37,39,40,52 On the other hand, SSc fibroblasts exhibit increased Smad7 and c-Ski/SnoN levels compared with normal fibroblasts in vivo and in vitro.32,55 This can be also explained by the stimulation of autocrine TGF-β signaling, because TGF-β1 can induce their expression. Although Smad7 constitutively forms a complex with the TGF-β receptors, the inhibitory effect of Smad7 on the promoter activity of human COL1A2 is completely impaired in SSc fibroblasts, as described above.32 Similarly, c-Ski/SnoN cannot compete with p300 in these cells.55 Taken together, multiple negative feedback systems of TGF-β signaling by Smad7 and c-Ski/SnoN as well as Smurf are impaired in SSc fibroblasts.
These factors may contribute to the establishment of autocrine TGF-β signaling in SSc fibroblasts. In addition, most of the cultured SSc fibroblasts are reported to be myofibroblasts that have the ability to express α-smooth muscle actin (α-SMA).108 Also, as described above, the basal protein expression of MMP-1 or TIMP-1 tends to be reduced or increased in SSc fibroblasts compared with fibroblasts of normal subjects, respectively. Because TGF-β1 can induce α-SMA expression and myofibroblast differentiation or can upregulate TIMP-1 expression and downregulate MMP-1 expression in normal fibroblasts,109 the autocrine TGF-β signaling hypothesis can also explain these features of SSc fibroblasts. Therefore, TGF-β is likely to play a key role in the pathogenesis of SSc.
Connective tissue growth factor (CTGF) is a 36–38-kDa cystein-rich peptide containing 349 amino acids. CTGF contains four conserved modules that resemble domains in other extracellular proteins, including an insulin-like growth factor binding domain (module I), a chordin-like cystein-rich domain (module II), a thrombospondin type 1 repeat (module III) and a C-terminal cystein-knot (module IV).110 They orchestrate many essential biological functions such as mitogenesis, chemotaxis, ECM production, apoptosis and angiogenesis, depending on cell types.
Connective tissue growth factor has also been regarded as an important cytokine involved in the pathogenesis of SSc. CTGF is induced by TGF-β1 and enhances fibroblast cell growth and ECM production as a downstream mediator of TGF-β1.7,111,112In vivo s.c. injection of TGF-β1 alone transiently induces skin fibrosis, whereas serial addition of CTGF after TGF-β1 causes persistent dermal fibrosis.113 On the other hand, anti-CTGF antibodies can reduce skin fibrosis induced by s.c. injection of TGF-β1.114 These studies indicate that a two-step process of fibrosis occurs in SSc skin: TGF-β1 induces fibrosis in the early stage and afterwards CTGF contributes to maintain the fibrosis.115,116 CTGF may modulate the Smad pathway indirectly through interaction with TGF-β, thereby increasing receptor binding and prolonging TGF-β signaling.7,110
In the SSc serum and SSc skin or in cultured SSc fibroblasts, constitutive overexpression of CTGF is found.117–119 Its expression appears to correlate with the degree of fibrosis.118 In normal adult fibroblasts, TGF-β1 potently induces CTGF at the level of transcription through a Smad binding element located within the proximal promoter.110 However, mutagenesis of the Smad binding site does not reduce the high level of CTGF promoter activity observed in SSc fibroblasts. Thus, the excess CTGF expression in SSc fibroblasts might be due to the autocrine TGF-β signaling, but independent of Smad.97,120 To note, recently, a polymorphism at the Sp1/Sp3 binding site of CTGF promoter region is reported to be responsible for the elevated expression of CTGF in SSc patients.121
IL-4 and IL-13
The gene for IL-4 is closely linked to the IL-13 gene,129 and IL-4 protein has been shown to have a 30% identity in the amino acid sequence to IL-13 protein.130 Furthermore, it has been suggested that IL-4 receptor and IL-13 receptor may share a common component that appears to function in signal transduction. Both IL-4 and IL-13 are shown to be elevated in the serum and the affected skin of SSc patients, compared with those of normal controls.131,132 Also, IL-4 protein expression is upregulated in SSc fibroblasts cultures.133
Interleukin-13 induces the expression of type I collagen protein to the same extent as IL-4 in human skin fibroblasts.134,135 However, IL-13-deficient mice are protected from fluorescein isothiocyanate-induced lung fibrosis because of impaired collagen synthesis by fibroblasts, whereas IL-4-deficient mice are not protected.136 IL-13 may play more important roles in the regulation of fibrosis in vivo than IL-4. On the other hand, TIMP-2 protein and mRNA expression is induced by IL-4 in a dose- and time-dependent manner, but not by TGF-β1, in normal dermal fibroblasts.137 Thus, IL-4 can potentially alter the dermal matrix metabolism by regulating TIMP-2.