Transforming growth factor β receptors (TGFβRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFβR expression.
Transforming growth factor β receptors (TGFβRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFβR expression.
The levels of expression of TGFβR type II (TGFβRII) messenger RNA (mRNA), with or without stimulation by epidermal growth factor (EGF), were evaluated by Northern blot analysis, and the protein levels were determined by immunoblotting. The transcription activity of the TGFβRII gene was examined with luciferase assays using the −1670/+35 TGFβRII promoter luciferase construct.
EGF up-regulates the expression of TGFβRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFβRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFβRII mRNA half-life. EGF-mediated induction of TGFβRII expression was inhibited by treatment of fibroblasts with the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002, and Akt inhibitor also blocked EGF-induced expression of TGFβRII. In addition, EGF induced TGFβRII promoter activity, and this induction was significantly blocked by wortmannin, LY294002, or Akt inhibitor. Cotransfection with a dominant-negative mutant of p85 (the regulatory component of PI 3-kinase) or Akt significantly reduced the induction of TGFβRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFβRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFβRII but did not show further up-regulation of TGFβRII expression by EGF.
These results indicate that EGF-mediated induction of TGFβRII expression occurs at the transcription level, does not require de novo protein synthesis, and involves the PI 3-kinase/Akt signaling pathway, and that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGFβRII in scleroderma fibroblasts.
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.
Wortmannin, LY294002, and Akt inhibitor were purchased from Calbiochem (La Jolla, CA). Recombinant human EGF was obtained from R&D Systems (Minneapolis, MN). Actinomycin D and cycloheximide were purchased from Sigma (St. Louis, MO). The luciferase assay kit was purchased from Promega (Madison, WI). Antibodies specific for TGFβRII were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for Akt and phospho-Akt (Ser473), and the Akt kinase assay kit were obtained from New England Biolabs (Beverly, MA). Anti–PI 3-kinase p85 antibodies and antiphosphotyrosine (4G10) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). FuGENE 6 was obtained from Roche Diagnostics (Indianapolis, IN).
Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 randomly selected patients with diffuse cutaneous SSc of <2 years' duration, and from the dorsal forearm of 10 healthy donors. All biopsy specimens were obtained after receiving informed consent and institutional approval. Primary explant cultures were established in 25-cm2 culture flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mML-glutamine, and 50 μg/ml gentamicin, as described previously (12, 13). Fibroblast cultures were maintained at 37°C in 95% air, 5% CO2 and were studied between the third and sixth subpassages.
Fibroblasts were washed with phosphate buffered saline (PBS) at 4°C and solubilized in lysis buffer containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride (PMSF), as described previously (41, 42). Proteins were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were incubated overnight with anti-TGFβRI (1:500 dilution) or anti-TGFβRII (1:500 dilution) antibodies, washed, and incubated with a secondary antibody against rabbit IgG for 60 minutes. After washing, visualization was performed using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan). For immunoblotting using antibodies against phospho-Akt, membranes were incubated with anti–phospho-Akt (Ser473) monoclonal antibody (1:1,000 dilution) overnight at 4°C. As a loading control, immunoblotting was also performed using antibodies against total Akt (1:1,000 dilution).
Total RNA was extracted using an acid guanidinium thiocyanate–phenol–chloroform method (41, 42). Poly(A)+ RNA was extracted from total RNA using oligotex-dT30 (Roche Diagnostics) and analyzed by Northern blotting as described previously (41, 42). Two micrograms of poly(A)+ RNA was subjected to electrophoresis on 1% agarose/formaldehyde gels and blotted onto nylon filters (Roche Diagnostics). The filters were ultraviolet cross-linked, prehybridized, and hybridized, and sequentially hybridized with DNA probes for GAPDH and RNA probes for TGFβRII. The membranes were then washed and exposed to x-ray film.
To prepare extracts of total cellular proteins, fibroblasts were washed with PBS at 4°C and solubilized in lysis buffer containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.1% SDS, 50 mM sodium fluoride, 1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Five hundred micrograms of total cellular protein was incubated with antibodies to the p85 subunit of PI 3-kinase overnight at 4°C, followed by 2 hours of incubation with protein A agarose (Gibco BRL, Grand Island, NY) at 4°C. After 3 washes in lysis buffer, the immunocomplexes were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, and incubated with antiphosphotyrosine antibody. The membrane was washed and then incubated for 60 minutes with a secondary antibody against mouse IgG. As a loading control, immunoblotting was also performed using antibodies against total p85 (1:1,000 dilution).
The activation of Akt was examined using an Akt kinase assay kit according to the manufacturer's instructions. In this experiment, fibroblasts were serum-starved for 18 hours and treated with EGF for the indicated times. The fibroblasts were then washed with ice-cold PBS and lysed. For the Akt kinase assay, 200-μl aliquots of the lysates were incubated with immobilized anti–Akt 1G1 monoclonal antibody (1:10 dilution) overnight at 4°C for immunoprecipitation. For kinase assays, the beads were then incubated with 200 μM of ATP and 2 μg of GSK3 fusion protein as a substrate for Akt. The reaction was terminated by addition of 25 μl of SDS sample buffer. The samples were boiled for 5 minutes, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with anti–phospho-GSK3α/β (Ser21/9) antibody (1:1,000 dilution) overnight at 4°C. The membranes were then washed and incubated for 60 minutes with a secondary antibody against rabbit IgG.
TGFβRII promoter (−1670/+35) fragment derived as a Kpn I/Hind III fragment from TGFβRII promoter luciferase construct, which was kindly provided by Dr. Seong-Jin Kim (National Cancer Institute, Bethesda, MD) (43), was inserted into the luciferase vector pA3Luc, which was kindly provided by Dr. William M. Wood (University of Colorado Health Sciences Center, Denver) (44), to construct TGFβRII promoter–pA3Luc. The pA3Luc vector includes a trimerized SV40 poly(A) termination site, which reduces transcription read-through (45) and does not contain AP-1–responsive vector sequences (46). The dominant-negative mutant form of Akt was kindly provided by Dr. Brian A. Hemmings (Friedrich Miescher Institute, Basel, Switzerland) (47, 48). Plasmids used in transient transfection assays were purified twice on CsCl gradients, as described previously (42). At least 2 different plasmid preparations were used for each experiment.
For each transfection, 3 μg of TGFβRII promoter–pA3Luc, 1 μg of β-galactosidase (β-gal) (transfection efficiency control), and FuGENE 6 were combined and added to cells. Transfected cells were treated for 18 hours with EGF in serum-free modified Eagle's medium before cell lysis in 50 μl of Reporter Lysis Buffer (Promega). Luciferase activity was normalized by cotransfected β-gal activity to correct for the transfection efficiency. All transfections were repeated at least 3 times.
Statistical analysis was performed using the Mann-Whitney U test. P values less than 0.05 were considered significant.
In the present study, we investigated the effects of EGF on the expression of TGFβRs in human dermal fibroblasts. First, we investigated the dose dependency of the effect of EGF on the expression of TGFβRs. Human dermal fibroblasts were cultured until they were confluent and then were incubated for an additional 24 hours under conditions of serum starvation. Cells were subsequently incubated for 24 hours with the indicated doses of EGF, prior to protein extraction. The TGFβRII protein level was elevated maximally (2.2-fold) by treatment with 20 ng/ml or 50 ng/ml of EGF but was less elevated in response to treatment with higher concentrations of EGF (100 ng/ml) (Figures 1A and B). In contrast, EGF did not change the TGFβRI protein level. In addition, the TGFβRII mRNA level was elevated maximally (2.5-fold) by treatment with 20 ng/ml of EGF (Figures 1C and D).
Next, to examine the time dependency of the effect of EGF on the expression of TGFβRs, cells were incubated in serum-free medium for the indicated times in the presence or absence of 20 ng/ml of EGF, which was added 6, 12, 24, or 48 hours prior to protein extraction. The TGFβRII protein level was slightly elevated after 6 hours and was markedly increased (2.4-fold) after 48 hours in comparison with the level in control cells (Figures 2A and B). However, EGF did not change the TGFβRI protein level.
To determine whether the EGF-mediated induction of TGFβRII protein expression was correlated with an increase of the mRNA level, human dermal fibroblasts were incubated in the presence or absence of 20 ng/ml of EGF under the same conditions, and mRNA expression was analyzed by Northern blotting. The TGFβRII mRNA level was elevated after stimulation with EGF for 6 hours, and was markedly increased (2.3-fold) after 12 hours in comparison with the level in control cells (Figures 2C and D). Thus, the effect of EGF on the TGFβRII protein level paralleled that on the mRNA level. These results suggest that the mRNA and protein levels of TGFβRII were up-regulated by EGF.
To determine whether the EGF-mediated up-regulation of TGFβRII requires protein synthesis, human dermal fibroblasts were incubated with cycloheximide before EGF treatment. The addition of cycloheximide alone did not cause cell death and did not significantly alter the basal TGFβRII mRNA level. EGF induced an increase in TGFβRII mRNA expression in the presence of cycloheximide, similar to that which occurred in the absence of cycloheximide (Figures 3A and B). Thus, EGF-mediated up-regulation of TGFβRII is independent of new protein synthesis.
Next, to determine whether the up-regulation of TGFβRII expression was attributable to increased RNA synthesis, the effects of EGF on newly transcribed RNA were examined by the treatment of human dermal fibroblasts with actinomycin D. The addition of actinomycin D alone did not cause cell death and slightly decreased the basal TGFβRII mRNA level. In cells that had been treated with actinomycin D, EGF did not induce an increase in TGFβRII mRNA expression, in contrast to the increase observed in the absence of actinomycin D (Figures 3A and B). These findings indicate that EGF may contribute to increased transcription of TGFβRII mRNA.
In addition, we wished to determine whether EGF increased the stability of TGFβRII mRNA. Human dermal fibroblasts were treated with EGF for 12 hours, and then transcription was terminated by the addition of actinomycin D. Following the inhibition of transcription, the loss of EGF-induced TGFβRII mRNA was rapid and was not significantly different from that observed in the untreated cells (Figure 3C). The failure of EGF to increase the half-life of TGFβRII mRNA suggests that EGF-mediated induction of TGFβRII expression is regulated at the level of transcription. Taken together, these results indicate that EGF up-regulates TGFβRII mRNA expression at the transcription level, and that this induction is independent of new protein synthesis.
Because the PI 3-kinase signaling pathway is involved in the signaling by EGF, we investigated whether PI 3-kinase activation is involved in EGF-mediated TGFβRII mRNA induction. Pretreatment of cells with wortmannin (Figures 4A and B) or LY294002 (Figures 4C and D) slightly reduced the basal expression of TGFβRII mRNA and blocked EGF-mediated up-regulation of TGFβRII mRNA in a dose-dependent manner. Next, we examined the role of Akt in EGF-mediated up-regulation of TGFβRII mRNA using Akt inhibitor. Akt inhibitor also inhibited the EGF-induced increase of TGFβRII mRNA (Figures 4E and F). Moreover, EGF-induced up-regulation of TGFβRII protein was also prevented by treatment with wortmannin, LY294002, or Akt inhibitor, as shown by immunoblotting (Figures 4G and H). These results indicate that the activity of PI 3-kinase/Akt is involved in EGF-mediated induction of TGFβRII expression.
PI 3-kinase is composed of the p110 catalytic subunit and the p85 regulatory subunit. The activity of PI 3-kinase is regulated through tyrosine phosphorylation of p85. We investigated whether EGF treatment induces p85 phosphorylation in human dermal fibroblasts. Immunoprecipitation using anti–PI 3-kinase p85 antibodies revealed a marked activation (4.9-fold) of p85 after 15 minutes of treatment with EGF, followed by a slow decrease in the cellular level of the phosphorylated p85 (Figures 5A and B). This result suggests that EGF induces p85 tyrosine phosphorylation in human dermal fibroblasts. Next, we investigated EGF-mediated Akt phosphorylation in human dermal fibroblasts. Immunoblotting using anti–phospho-Akt (Ser 473) antibody revealed marked phosphorylation (4.9-fold) of Akt after 5 minutes of treatment with EGF, and this increase was sustained for 30 minutes (Figures 5C and D). Specific inhibitors of PI 3-kinase, wortmannin and LY294002, inhibited PI 3-kinase–induced phosphorylation of Akt. Immunoblotting for total Akt protein demonstrated that the amount of Akt did not significantly change in the presence of EGF.
Cells were pretreated with wortmannin, LY294002, or a specific inhibitor of Akt, Akt inhibitor, for 1 hour prior to stimulation with EGF, and then an Akt kinase assay was performed. The results of the Akt kinase assay showed that EGF stimulation increased Akt kinase activity. The level of phosphorylated GSK3β was maximal (4.7-fold) after 15 minutes of incubation with EGF (Figures 5E and F). Wortmannin (100 nM), LY294002 (30 μM), or Akt inhibitor (20 μM) significantly inhibited the Akt-induced phosphorylation of GSK3β (Figures 5E and F). These results suggest that treatment with EGF results in activation of the PI 3-kinase/Akt signaling pathway in human dermal fibroblasts, and that wortmannin, LY294002, and Akt inhibitor inhibit activation of the PI 3-kinase/Akt signaling pathway in human dermal fibroblasts stimulated by EGF.
Next, we used the TGFβRII promoter–pA3Luc (−1670/+35) to determine whether EGF induced the TGFβRII gene at the transcription level. The TGFβRII promoter activity was elevated maximally (2.5-fold) by treatment with 20 ng/ml of EGF (Figure 6). To investigate the role of the PI 3-kinase/Akt signaling pathway in the transcriptional regulation of TGFβRII, we examined the TGFβRII promoter activity in human dermal fibroblasts treated with pharmacologic reagents. Wortmannin, LY294002, or Akt inhibitor significantly blocked the EGF-mediated TGFβRII promoter activity (Figure 7). We then examined whether EGF-induced TGFβRII promoter activity was simultaneously inhibited by these inhibitors. LY294002 plus Akt inhibitor, as well as wortmannin plus Akt inhibitor, did not exhibit synergistic inhibitory effects on EGF-mediated induction (Figure 7). We also examined the participation of Ras/Raf/MEK/ERK signaling in EGF-mediated TGFβRII up-regulation. We analyzed the effect of the MEK inhibitor, PD98059, on EGF-induced TGFβRII promoter activity and observed that PD98059 could not inhibit the EGF-mediated induction of TGFβRII promoter activity (Figure 7). This result indicates that Ras/Raf/MEK/ERK signaling may not participate in EGF-mediated TGFβRII up-regulation. This finding suggests the importance of the PI 3-kinase/Akt signaling pathway in mediating the induction of TGFβRII transcription activity by EGF.
To further examine whether PI 3-kinase was required for TGFβRII promoter activity, a dominant-negative mutant of p85 (a regulatory component of PI 3-kinase) was transiently transfected into human dermal fibroblasts. Overexpression of the dominant-negative p85 mutant in human dermal fibroblasts suppressed the induction of TGFβRII promoter activity by EGF (Figure 8). Moreover, overexpression of the constitutive active p110 (a catalytic component of PI 3-kinase) mutant into human dermal fibroblasts induced TGFβRII promoter activity. Because PI 3-kinase activates the downstream effector Akt, we examined whether Akt affects TGFβRII promoter activity. Expression of a dominant-negative mutant of Akt attenuated the induction of TGFβRII promoter activity by EGF to the same extent as did the dominant-negative mutant of p85 (Figure 9). These results suggest that the PI 3-kinase/Akt signaling pathway participates in the regulation of TGFβRII promoter activity.
The dermal fibroblast activation in scleroderma may be a result of stimulation by autocrine TGFβ. This notion is supported by our recent finding that SSc fibroblasts express elevated levels of TGFβRI and TGFβRII, and that this correlates with elevated levels of collagen α2(I) (12, 49–51). However, the mechanism of up-regulation of TGFβRI and TGFβRII remains unknown. In the present study, we showed that EGF up-regulated TGFβRII protein levels, and we examined the effect of EGF on up-regulated TGFβRII expression in scleroderma fibroblasts. Scleroderma fibroblasts expressed increased levels of TGFβRII. In contrast, EGF did not induce further up-regulation of TGFβRII expression in scleroderma fibroblasts, whereas EGF up-regulated TGFβRII expression in human dermal fibroblasts (Figure 10). This result suggests that some EGF-activated signaling pathways, including PI 3-kinase/Akt, have already been activated in scleroderma fibroblasts.
To investigate whether up-regulation of the TGFβRII level in scleroderma fibroblasts might be mediated via the PI 3-kinase/Akt signaling pathway, we used wortmannin, LY294002, and Akt inhibitor. These inhibitors decreased up-regulated TGFβRII expression in scleroderma fibroblasts in a dose-dependent manner (Figure 11). These results indicate that the PI 3-kinase/Akt signaling pathway may contribute to the up-regulation of TGFβRII expression in scleroderma fibroblasts and to the resistance of scleroderma fibroblasts to the effect of EGF on TGFβRII expression.
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.
We thank Dr. S. J. Kim for kindly providing TGFβRII promoter luciferase constructs. We thank Dr. W. M. Wood for kindly providing pA3Luc luciferase constructs. We thank Dr. B. A. Hemmings for kindly providing dominant-negative constructs of Akt.