Transforming growth factor β (TGFβ) is a multifunctional protein that plays an important role in regulating cellular growth, differentiation, adhesion, and apoptosis in many biologic systems (1–3). TGFβ inhibits the growth of most cell types. In addition, TGFβ causes the deposition of extracellular matrix (ECM) by simultaneously stimulating dermal fibroblasts to increase the production of ECM proteins such as collagen, fibronectin, or proteoglycan, decrease the production of matrix-degrading proteases, increase the production of inhibitors of these proteases, and modulate the expression of integrins (2, 3). TGFβ binds to transmembrane receptors that have intrinsic serine/threonine kinase activity (4). TGFβ receptor type II (TGFβRII) binds TGFβ, and then the TGFβRI is recruited into a heteromeric complex. TGFβRII transphosphorylates the glycine/serine-rich domain of TGFβRI kinase (5). Following the phosphorylation of SMAD-2 or SMAD-3 by the activated TGFβRI, a heteromeric complex is formed with SMAD-4, resulting in translocation of the complex into the nucleus (6, 7). This complex can act directly as a transcription activator and can also indirectly regulate gene transcription by interacting with other transcription factors (8–11).
A critical mechanism for regulating the cellular response to cytokines and hormones resides at the level of receptor expression. Modulation of the level of TGFβRI and TGFβRII expression plays an important role in both the mechanism of wound healing and the progression of malignancy. Disorders of TGFβR expression lead to various diseases. For example, up-regulation of TGFβR expression has been demonstrated in fibrotic diseases such as systemic sclerosis (SSc; scleroderma), localized scleroderma, hepatic fibrosis, idiopathic hypertrophic obstructive cardiomyopathy, and atherosclerosis (12–16). Up-regulation of TGFβR expression results in the deposition of ECM components. In contrast, reduction of TGFβR levels contributes to the resistance of tumor cells to TGFβ. Several lines of evidence suggest that transcriptional repression of the TGFβR gene may be a major mechanism to inactivate TGFβR in tumor cells (17).
Epidermal growth factor (EGF) is a key regulatory component of cell growth and differentiation in a variety of cell types (18). In human dermal fibroblasts, EGF is both motogenic and mitogenic. EGF signaling occurs predominantly through binding to the EGF receptor and its dimerization partner ErbB-2. Autophosphorylation of activated EGF receptors stimulates a number of signal transduction pathways, including the Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (MEK/ERK) pathway and the phosphoinositide 3-kinase (PI 3-kinase)/Akt pathway. It is becoming increasingly clear that Akt has a pivotal role in cell cycle progression (19–22), angiogenesis (23), inhibition of apoptosis (24–29), and cell growth (30). The mechanisms by which Akt exerts its antiapoptotic effect in cells have attracted much attention. Targets of Akt related to apoptosis include the Bcl-xL/Bcl-2–associated death promoter (BAD) (31, 32), human caspase 9 (33), forkhead transcription factors (FKHR, FKHR-L1, and AFX) (34–37), nuclear factor κB (NF-κB) (27, 38), glycogen synthase kinase 3β (GSK3β) (39), and cAMP response element binding protein (CREB) (40).
For regulation of tissue homeostasis, the balance of EGF and TGFβ signaling in human dermal fibroblasts seems to be critical. Furthermore, clarifying the mechanism of the regulation of TGFβR expression in normal human dermal fibroblasts should be instructive for elucidating the pathogenesis of the progression of fibrotic diseases or malignancy. TGFβ signaling is initiated by binding of TGFβ to TGFβRII, and cancer cells, in which TGFβRII is repressed, are resistant to TGFβ. Thus, TGFβRII plays a critical role in receptor activation and subsequent signal propagation, functioning both to bind ligand and to activate TGFβRI. In the present study, we examined the regulation of TGFβRII expression in human dermal fibroblasts by EGF and the contribution of the PI 3-kinase/Akt signaling pathway to the EGF-mediated regulation of TGFβRII expression. In addition, we investigated the effect of EGF on up-regulated TGFβRII expression in scleroderma fibroblasts.
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A complex network of cytokines and growth factors orchestrates cell proliferation, differentiation, and wound healing in the skin. Various stimuli activate divergent signaling pathways and induce distinct cellular responses. Among these stimuli, TGFβ signaling plays a critical role in controlling cellular growth and ECM production. Repression of the expression of TGFβRs occurs in various types of human cancer cells, while up-regulation of the expression of TGFβRs occurs in fibrotic disorders. The expression of TGFβRs is regulated by a plethora of external factors, including cytokines and growth factors. It has been shown by Northern blot analysis and binding studies that 1,25-dihydroxyvitamin D3 and prostaglandin E2 down-regulate TGFβRII expression in human osteoblastic cells and human fibroblasts, respectively (52, 53). Furthermore, binding studies revealed down-regulation of TGFβRI expression by interferon-γ in human monocytes (54). In human lung fibroblasts (55) and human corpus carvernosum smooth muscle cells (56), TGFβ1 increases the steady-state level of TGFβRI mRNA, possibly by increasing TGFβRI promoter activity (55).
Recently, it was reported that in COLO-357 pancreatic cancer cells, TGFβRI and TGFβRII mRNA and protein levels are up-regulated by TGFβ1 (57). Regarding the effects of integrins, the interaction of α2β1 integrin with type I collagen down-regulates TGFβRs (58), whereas α5β1 integrin binding to fibronectin up-regulates TGFβRII (59). Although the modulation of TGFβR expression by many cytokines and hormones has been demonstrated, the exact mechanisms involved in the regulation of the expression of TGFβRs by growth factors remain unclear.
To our knowledge, this report is the first to describe involvement of the PI 3-kinase/Akt signaling pathway in the induction of EGF-mediated TGFβRII expression. In the present study, we demonstrated that the PI 3-kinase/Akt signaling pathway is a required signaling intermediate for EGF-induced up-regulation of TGFβRII expression, as indicated by the fact that wortmannin, LY294002, and Akt inhibitor blocked the increase in TGFβRII mRNA expression and the appearance of TGFβRII protein following EGF treatment. In addition, our results indicate that the EGF-induced increase in mRNA expression was controlled at the level of transcription. Blocking mRNA synthesis with actinomycin D completely abrogated the EGF-induced TGFβRII up-regulation. Induction of TGFβRII mRNA by EGF was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for this response. Moreover, treatment with EGF did not significantly change the TGFβRII mRNA half-life.
These results suggest that regulation at the level of transcription is critical for EGF-mediated induction of TGFβRII expression in human dermal fibroblasts. Furthermore, the pretreatment of cells with wortmannin, LY294002, or Akt inhibitor significantly attenuated the EGF-induced enhancement of TGFβRII promoter activity. Our observations that EGF-mediated induction of TGFβRII promoter activity is inhibited by expression of a dominant-negative form of p85 or Akt and is activated by a constitutive active form of p110 indicate that the PI 3-kinase/Akt signaling pathway appears to participate in the regulation of TGFβRII promoter activity. These results suggest that the PI 3-kinase/Akt signaling pathway is critical in the regulation of TGFβRII expression by EGF in human dermal fibroblasts.
In addition, we found that PI 3-kinase and Akt inhibitors decreased overexpression of TGFβRII in scleroderma fibroblasts. This result indicates that PI 3-kinase and/or Akt might induce up-regulation of TGFβRII in scleroderma fibroblasts. In addition, we used Western blotting to examine the expression levels and activities of PI 3-kinase/Akt in scleroderma fibroblasts. However, no significant difference between normal fibroblasts and scleroderma fibroblasts was observed (results not shown). This lack of difference might be attributable to the low sensitivity of our experimental methods or the participation of other signaling pathways. We need to clarify this point in a future study.
PI 3-kinase/Akt is activated in response to a variety of stimuli, including growth factors and cytokines (60). Other studies have shown that the PI 3-kinase/Akt signaling pathway can regulate the expression of various genes at the transcription level. Akt increases the expression of glucose transporter 1 protein in hepatoma cells (61), Bcl-2 in PC12 cells (62), and vascular endothelial growth factor in NIH3T3 fibroblasts (63). Akt phosphorylates a number of downstream molecules, including BAD, NF-κB, CREB, GSK3β, and a member of the FKHR family. Choi et al demonstrated that CREB controls the expression of TGFβRII (43). Whether or not CREB plays a role in EGF-mediated up-regulation of TGFβRII at the level of transcription remains to be elucidated. The roles of the downstream signaling cascade of the PI 3-kinase/Akt signaling pathway and of crosstalk between these pathways and other signaling pathways in the EGF-mediated induction of expression need to be examined.
The accumulation of ECM in tissues is the chief pathologic feature of fibrotic disorders. TGFβ signaling has been implicated in the primary pathogenesis of fibrosis (2). In the case of scleroderma, in which progressive fibrosis in the skin is a major cause of disease, it was reported that scleroderma fibroblasts of the involved area did not secrete increased levels of TGFβ1 (64). The mechanism of tissue fibrosis in such diseases remains to be determined. We previously reported the overexpression of TGFβRI and TGFβRII in scleroderma fibroblasts compared with normal human dermal fibroblasts, indicating one possible mechanism of autocrine control of TGFβ activity by overexpression of TGFβRI or TGFβRII (12). Furthermore, cotransfection of TGFβRI and TGFβRII expression vectors and a collagen α2(I) promoter/chloramphenicol acetyltransferase reporter gene showed that increasing the TGFβR level induced a 3–4–fold increase of collagen promoter activity, and this increase was sensitive to anti-TGFβ1 antibody (12). In addition, we reported that SSc fibroblasts secreted amounts of TGFβ similar to those secreted by control fibroblasts, and that the blockade of TGFβ signaling with anti-TGFβ antibodies or a TGFβ1 antisense oligonucleotide abolished the increased mRNA expression, as well as the up-regulated transcription activity of the human collagen α2(I) gene in SSc fibroblasts (49).
In idiopathic hypertrophic obstructive cardiomyopathy, which is characterized by regional myocardial hypertrophy with marked cardiomyocyte hypertrophy and a significant increase of the ECM, TGFβRs are overexpressed on cardiomyocytes and fibroblasts (15). In addition, with regard to the EGF receptor, it has been demonstrated that a 2-fold increase in receptor expression led to at least a 10-fold decrease in the concentration of ligand required for induction of a biologic response (65). These findings suggest that autocrine regulation of TGFβ activity might result from receptor up-regulation rather than an increase of ligand. In addition, resistance of scleroderma fibroblasts to the EGF effect on TGFβRII expression indicated that some abnormal signaling pathways have up-regulated TGFβRII expression, and EGF did not induce further up-regulation of TGFβRII expression in scleroderma fibroblasts. In future study, the abnormal signaling pathways in scleroderma fibroblasts need to be clarified.
Recently, adenovirus-mediated local expression of a dominant-negative TGFβRII and infusion of soluble TGFβRII have been demonstrated to be effective for prevention of hepatic fibrosis (66, 67). Taken together, these results suggest that, in fibrotic disorders, TGFβ signaling may play a central role, and that the mechanism of regulation of TGFβRI and TGFβRII in such diseases may be critical. Moreover, our results indicate that the PI 3-kinase/Akt signaling pathway has a significant relationship to the modulation of TGFβRII, and that blockade of the PI 3-kinase/Akt signaling pathway may also have therapeutic value.
In conclusion, we showed that EGF up-regulates TGFβRII expression at the transcription level, and we demonstrated for the first time that a PI 3-kinase/Akt signaling pathway is essential for the EGF-mediated induction of TGFβRII expression. Scleroderma fibroblasts did not show further up-regulation of TGFβRII expression by EGF, and PI 3-kinase/Akt inhibitors decreased up-regulated expression of TGFβRII in scleroderma fibroblasts. These results indicate that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in up-regulation of TGFβRII in scleroderma fibroblasts.