Increased transcriptional activities of transforming growth factor β receptors in scleroderma fibroblasts

Authors


Abstract

Objective

To investigate the molecular mechanism of the overexpression of transforming growth factor β receptors (TGFβRs) in dermal fibroblasts from patients with systemic sclerosis (SSc).

Methods

Dermal fibroblasts from 7 patients with diffuse SSc of recent onset and from 7 healthy individuals were studied. The expression of TGFβR type I (TGFβRI), TGFβRII, and type I collagen proteins in dermal fibroblasts was determined by immunoblotting. TGFβRI, TGFβRII, and α2(I) collagen messenger RNA (mRNA) were evaluated by Northern blot analysis. The transcriptional activities of the TGFβRI and TGFβRII genes were examined by luciferase assay.

Results

SSc fibroblasts expressed increased levels of TGFβRI and TGFβRII protein and mRNA, as well as increased levels of type I collagen protein and α2(I) collagen mRNA. Moreover, the half-lives of TGFβRI and TGFβRII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGFβRI and TGFβRII genes were both significantly increased in SSc fibroblasts compared with those in control fibroblasts. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited TGFβRI promoter activity in SSc fibroblasts, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), inhibited TGFβRII promoter activity in SSc fibroblasts. Moreover, calphostin C and LY294002 inhibited the up-regulation of TGFβRI and TGFβRII mRNA, respectively, in SSc fibroblasts.

Conclusion

These results suggest that increased levels of TGFβRs in SSc fibroblasts play a role in excessive collagen production, and that up-regulation of TGFβR expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGFβR expression in SSc fibroblasts.

Excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc; scleroderma) (1). The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that activation of lesional fibroblasts contributes to the fibrotic process (2, 3). Numerous differences between cultured SSc and healthy skin fibroblasts that can contribute to excessive ECM deposition in vivo have been demonstrated, such as elevated expression of types I, III, VI, and VII collagen, fibronectin, and glycosaminoglycans (3–10).

The mechanism of dermal fibroblast activation in SSc is currently unknown. However, many of the characteristics of SSc fibroblasts resemble those of healthy fibroblasts stimulated by transforming growth factor β (TGFβ) (11), suggesting that the dermal fibroblast activation in SSc may be a result of stimulation by autocrine TGFβ. This notion is supported by the recent finding that SSc fibroblasts express elevated levels of TGFβ receptor type I (TGFβRI) and TGFβRII messenger RNA (mRNA), and that this expression correlates with elevated levels of α2(I) collagen mRNA (12). In addition, we have previously 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 with a TGFβ1 antisense oligonucleotide abolished the increased mRNA expression, as well as the up-regulated transcriptional activity, of the human α2(I) collagen gene in SSc fibroblasts (13).

Clarifying the mechanism of up-regulated TGFβR expression in SSc fibroblasts could be instructive for elucidating the pathogenesis of progression of fibrotic diseases. The present study was undertaken to investigate the mechanism of up-regulation of TGFβRI and TGFβRII in SSc fibroblasts. First, we investigated the levels of TGFβRs and type I collagen proteins in SSc and control fibroblasts. We then investigated TGFβRI, TGFβRII, and α2(I) collagen mRNA expression, the half-lives and transcriptional activities of the genes, and the involved signaling pathways.

MATERIALS AND METHODS

Reagents

Actinomycin D was purchased from Sigma (St. Louis, MO). The luciferase assay kit was purchased from Promega (Madison, WI). Antibody for type I collagen was obtained from Southern Biotechnology (Birmingham, AL). Antibodies specific for TGFβRI and TGFβRII were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059, LY294002, and calphostin C were purchased from Calbiochem (La Jolla, CA).

Cell cultures

Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 7 randomly selected patients with diffuse cutaneous SSc of <2 years' duration and from the dorsal forearm of 7 healthy donors. All biopsy samples were obtained with informed consent and institutional approval. Primary explant cultures were established in 25-cm2 culture flasks in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 50 μg/ml gentamycin, as described previously (12, 13). Monolayer cultures were maintained at 37°C in 5% CO2 in air. Fibroblasts between the third and sixth subpassages were used for experiments.

Cell lysis and immunoblotting

Fibroblasts were washed with phosphate buffered saline 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, as described previously (13). The lysates were incubated for 30 minutes at 4°C and were then centrifuged for 15 minutes at 4°C. Protein concentrations of lysates were determined using Bio-Rad (Hercules, CA) protein assay reagent. Proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated overnight with anti-TGFβRI (50 ng/ml) or anti-TGFβRII (50 ng/ml) antibodies, washed, and incubated with a secondary antibody against rabbit IgG for 60 minutes. After washing, visualization was performed by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's recommendations. The densities of bands were measured with a densitometer.

RNA preparation and Northern blot analysis

Fibroblasts were grown to 80% confluence in MEM supplemented with 10% FBS and were then incubated for 24 hours in serum-free medium, as described previously (12, 13). Total RNA was extracted using an acid guanidinium thiocyanate–phenol–chloroform method (12, 13). Poly(A)+ RNA was extracted from total RNA using an oligotex-dT30 <Super>kit (Takara Shuzo, Shiga, Japan) and analyzed by Northern blotting as described previously (12, 13). Poly(A)+ RNA (2 μg) was subjected to electrophoresis on 1% agarose/formaldehyde gels and blotted onto nylon filters (Roche Diagnostics, Indianapolis, IN). The filters were ultraviolet cross-linked, prehybridized, and hybridized, and were then sequentially hybridized with DNA probes for α2(I) procollagen and GAPDH or with RNA probes for TGFβRI and TGFβRII. The membrane was then washed and exposed to x-ray film.

Plasmids

TGFβRI promoter luciferase constructs (−726 to −108) were kindly provided by Dr. Ronald H. Goldstein (Boston, MA) (14). TGFβRII promoter luciferase constructs (−1670 to +35) were kindly provided by Dr. Seong-Jin Kim (National Cancer Institute, Bethesda, MD) (15). Plasmids used in transient transfection assays were purified twice on CsCl gradients, as described previously (12, 13). At least 2 different plasmid preparations were used for each experiment.

Transient transfection and luciferase assays

For each transfection, 1 μg of TGFβRI or TGFβRII promoter luciferase constructs, 1 μg of β-galactosidase (transfection efficiency control), and 3 μl of FuGENE 6 (Roche Diagnostics, Indianapolis, IN) were combined and added to cells. Cells were harvested 48 hours after addition of the DNA, and extracts were assayed for luciferase activity. Luciferase activity was normalized to cotransfected β-galactosidase activity to correct for transfection efficiency. All transfections were repeated at least 3 times.

RESULTS

Increased expression of type I collagen, TGFβRI, and TGFβRII in SSc dermal fibroblasts

SSc fibroblasts were reported to overexpress type I collagen mRNA and protein. In order to investigate the role of TGFβRs in up-regulated type I collagen expression in SSc fibroblasts, we examined the expression of TGFβRI, TGFβRII, and α2(I) collagen mRNA in SSc and control fibroblasts (Figures 1A and B). SSc fibroblasts expressed increased levels of both TGFβRI and TGFβRII mRNA as well as correlated increased levels of α2(I) collagen mRNA. In addition, we examined the correlation between type I collagen and TGFβR protein levels in SSc and control fibroblasts (Figures 1C and D). SSc fibroblasts expressed increased levels of both TGFβRI and TGFβRII proteins as well as correlated increased levels of type I collagen protein. These results indicate that SSc fibroblasts overexpressed both TGFβRI and TGFβRII mRNA and protein, and that these increases correlated positively with elevated expression of α2(I) collagen mRNA and type I collagen protein.

Figure 1.

Correlation of increased expression of transforming growth factor β receptor type I (TβRI) and TβRII with increased expression of α2(I) collagen in scleroderma (systemic sclerosis; SSc) fibroblasts.A, Northern blot analysis of TβRI, TβRII, and α2(I) collagen mRNA expression was performed using 2 μg of Poly(A)+ RNA. Levels of GAPDH mRNA are shown as a loading control. B, TβRI, TβRII, and α2(I) collagen mRNA levels were quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples. Values from SSc fibroblasts indicate band densities relative to that of control fibroblasts, which was set at 1. Shown are the mean and SD resulting from quantitative analysis of 7 paired samples of SSc and control fibroblasts.C, Cell lysates (15 μg protein/sample) were subjected to immunoblotting with anti-TβRI, anti-TβRII, and anti–type I collagen antibodies. Levels of β-actin are shown as a loading control. D, TβRI, TβRII, and type I collagen protein levels quantitated by scanning densitometry are shown relative to that of control fibroblasts, which was set at 1. Shown are the mean and SD resulting from quantitative analysis of 7 paired samples of SSc and control fibroblasts. ∗ = P < 0.05; ∗∗ = P < 0.01, versus control fibroblasts, by Mann-Whitney test for comparison of means. NS = normal samples.

Mechanisms of up-regulation of TGFβRI and TGFβRII

The steady-state level of mRNA can be affected by the level of gene transcription and/or the stability of mRNA. To investigate whether up-regulation of TGFβRI and TGFβRII expression was due to increased message stability, the half-lives of the TGFβRI and TGFβRII genes were examined by the treatment of SSc or control fibroblasts with actinomycin D. After treatment with actinomycin D, there were no significant differences in the half-lives of TGFβRI and TGFβRII mRNA between SSc and control fibroblasts (Figures 2A and B).

Figure 2.

Effects of actinomycin D on TβRI and TβRII up-regulation. Human dermal fibroblasts were serum starved for 24 hours and incubated in the absence or presence of 2.5 μg/ml of actinomycin D for the indicated periods of time. Poly(A)+ RNA was extracted from the cells at the indicated times after actinomycin D administration. Northern blot analysis of TβRI and TβRII mRNA expression was performed using 2 μg of poly(A)+ RNA. The corrected density of A, TβRI or B, TβRII was expressed as a percentage of the value at hour 0 and plotted on a logarithmic scale. Representative results shown are from 1 of 3 independent experiments. See Figure 1 for definitions.

Our findings indicate that the increasing stability of TGFβRI or TGFβRII might not result in up-regulation of TGFβRI or TGFβRII gene expression. To investigate the role of transcriptional regulation of TGFβRI and TGFβRII genes, we next examined the promoter activity using the luciferase reporter gene plasmid constructs under the transcriptional control of the TGFβRI and TGFβRII promoters. The TGFβRI promoter lacks TATA and CAAT boxes, but is highly GC-rich and contains putative Sp1 binding sites (14). The TGFβRII promoter lacks TATA and CAAT boxes, but contains putative Sp1 sites and a potential activator protein 1 site or potential cAMP response element/activating transcription factor sites (15).

SSc or control dermal fibroblasts were transfected with either TGFβRI or TGFβRII promoter reporter gene plasmid construct. The cells were then lysed, and promoter-mediated luciferase activities were determined. TGFβRI and TGFβRII promoter activities were both significantly up-regulated in SSc fibroblasts compared with control fibroblasts (Figure 3). These results suggest that transcriptional activation causes the up-regulation of the steady-state levels of TGFβRI and TGFβRII mRNA.

Figure 3.

Up-regulation of TβRI and TβRII promoter activity. Seven normal control fibroblasts or 7 SSc fibroblasts were transiently transfected with the TβRI promoter construct or the TβRII promoter construct. Values represent the TβRI or TβRII promoter activities relative to that of control fibroblasts, which was set at 1. Data are expressed as the mean and SD of 5 experiments. ∗ = P < 0.05; ∗∗ = P < 0.01, versus control fibroblasts, by Mann-Whitney test for comparison of means. See Figure 1 for definitions.

Blocking of up-regulated TGFβR expression by inhibitors of intracellular signaling pathways

Recently, Czuwara-Ladykowska et al have reported that platelet-derived growth factor (PDGF) up-regulates the mRNA levels of TGFβRI and TGFβRII through the mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (MEK/ERK) pathway (16). PDGF was known to activate various signaling pathways (e.g., phosphoinositide 3-kinase [PI 3-kinase], protein kinase C [PKC], and MEK/ERK pathways).

In order to investigate which intracellular signaling pathway plays a crucial role in transcriptional activation of TGFβR genes in SSc fibroblasts, we examined the effect of inhibitors of intracellular signaling, such as PI 3-kinase, PKC, and MEK/ERK, on the promoter activity of TGFβRI and TGFβRII. As shown in Figure 4A, in SSc fibroblasts, TGFβRI promoter activity was inhibited by calphostin C, an inhibitor of PKC signaling pathways, and TGFβRII promoter activity was inhibited by LY294002, an inhibitor of PI 3-kinase. We then investigated the effect of these pharmacologic reagents on TGFβR mRNA levels by Northern blotting. Treatment of control dermal fibroblasts with the inhibitors did not change the basal mRNA expression of TGFβRs (Figures 4B and C). Pretreatment of SSc fibroblasts with calphostin C reduced the expression of TGFβRI mRNA, and LY294002 blocked up-regulation of TGFβRII mRNA in SSc fibroblasts (Figures 4D and E). In contrast, PD98059 slightly increased the expression of TGFβRII mRNA. These results suggest that PKC or PI 3-kinase signaling pathways might contribute to the up-regulated promoter activity of the TGFβRI or TGFβRII gene, respectively, and that this signaling pathway–induced transcriptional activation of TGFβRs might play an important role in up-regulation of TGFβRI and TGFβRII mRNA.

Figure 4.

Blocking of TβR promoter activity by inhibitors of intracellular signaling pathways. A, SSc fibroblasts were transiently transfected with the TβRI or TβRII promoter luciferase construct along with vehicle (DMSO) alone (control), 30 μM LY294002, 200 nM calphostin C, or 30 μM PD98059. Values represent the TβRI or TβRII promoter activities relative to that of SSc fibroblasts treated with vehicle, which was set at 1. Data are expressed as the mean and SD of 5 experiments. ∗ = P < 0.05 versus SSc fibroblasts treated with vehicle, by Mann-Whitney test for comparison of means. B, Normal control fibroblasts were serum starved for 24 hours and treated with vehicle alone (control), 30 μM LY294002, 200 nM calphostin C, or 30 μM PD98059 for 24 hours. Northern blot analysis of TβRI and TβRII mRNA expression was performed using 2 μg of poly(A)+ RNA. Levels of GAPDH mRNA are shown as a loading control. C, TβRI and TβRII mRNA levels were quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples. D, SSc fibroblasts were serum starved for 24 hours and treated with vehicle alone (control), 30 μM LY294002, 200 nM calphostin C, or 30 μM PD98059 for 24 hours. Northern blot analysis of TβRI and TβRII mRNA expression was performed using 2 μg of poly(A)+ RNA. Levels of GAPDH mRNA are shown as a loading control. E, TβRI and TβRII mRNA levels were quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples. In A, C, and E, open bars indicate TβRI levels and solid bars indicate TβRII levels. Representative results shown are from 1 of 3 independent experiments. See Figure 1 for definitions.

DISCUSSION

A critical mechanism for regulating the cellular response to cytokines and hormones resides at the level of receptor expression. The modulation of the level of TGFβRI and TGFβRII expression plays important roles, both in the mechanism of wound healing and in the progression of fibrotic diseases or malignancy. The dysregulation of TGFβR expression leads to various diseases. For example, up-regulation of TGFβR expression was demonstrated in fibrotic diseases, such as SSc, localized scleroderma, idiopathic hypertrophic obstructive cardiomyopathy, hepatic fibrosis, and atherosclerosis (12, 13, 16–20). Up-regulation of TGFβR expression may establish an autocrine TGFβ loop and result in the deposition of ECM component. In several cancer cells, the loss of sensitivity to TGFβ has been derived from the loss of expression of TGFβRs, and the transcriptional repression of TGFβRs is a frequent cause of this deficiency (21). However, the mechanism of the overexpression of TGFβRs in fibrotic diseases remains to be determined.

In the present study, we have demonstrated that SSc fibroblasts expressed increased levels of TGFβRI and TGFβRII protein and mRNA, as well as increased levels of type I collagen protein and α2(I) collagen mRNA. Moreover, the half-lives of TGFβRI and TGFβRII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGFβRI and TGFβRII genes significantly increased in SSc fibroblasts compared with control fibroblasts.

Although various cytokines or growth factors have been reported to regulate the expression of TGFβRs, little is known about signaling pathways regulating TGFβR expression. In our study, blocking signaling pathways using pharmacologic reagents significantly abrogated TGFβRI or TGFβRII transcriptional activation and decreased their mRNA levels. The fact that TGFβRI and TGFβRII promoter activities are down-regulated by calphostin C and LY294002, respectively, suggests that PKC and/or PI 3-kinase signaling pathways might contribute to the transcriptional activation of these receptors. In contrast, PD98059 slightly increased the level of TGFβRII mRNA (see Figures 4D and E). However, PD98059 did not increase levels of TGFβRII mRNA in other SSc fibroblasts. In contrast, LY294002 did decrease levels of TGFβRII mRNA in other SSc fibroblasts. We think that the PI 3-kinase signaling pathway may play a more important role in the up-regulation of TGFβRII levels.

Taken together, our results indicate that the PKC and/or PI 3-kinase signaling pathways are significantly related to the modulation of TGFβRI and/or TGFβRII, respectively, and that the blockade of the PKC and/or PI 3-kinase signaling pathways may also have therapeutic value. Tourkina et al reported that overexpression of tenascin C results from abnormal PKCε activity in SSc fibroblasts (22). In addition, Jimenez et al showed that overexpression of PKCδ in SSc fibroblasts leads to increased transcriptional activity of α2(I) collagen (23). Future studies should identify abnormal signaling pathways and determine their influence on ECM production in SSc fibroblasts.

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. With respect to SSc, in which progressive fibrosis in the skin is a major cause of disease, it was reported that SSc fibroblasts of the involved area did not secrete increased levels of TGFβ1 (13, 24). The mechanism of tissue fibrosis in such diseases remains to be determined. Investigators in our group have reported the overexpression of TGFβRI and TGFβRII in SSc fibroblasts compared with normal human dermal fibroblasts, indicating one possible mechanism of autocrine TGFβ activity by overexpression of TGFβRI or TGFβRII (12). Furthermore, cotransfection of TGFβRI and TGFβRII expression vector and an α2(I) collagen promoter/chloramphenicol acetyltransferase reporter gene showed that increasing TGFβR levels induced a 3–4-fold increase of collagen promoter activity, and that this was sensitive to anti-TGFβ1 antibody (12).

Regarding idiopathic hypertrophic obstructive cardiomyopathy, which is characterized by regional myocardial hypertrophy with marked cardiomyocyte hypertrophy and a significant increase in ECM, TGFβRs are overexpressed on cardiomyocytes and fibroblasts (18). Other investigators have reported that transient overexpression of TGFβRs carrying a heteromeric combination of cytoplasmic domains resulted in ligand-independent responses (25). In addition, with regard to the epidermal growth factor receptor, it has been demonstrated that a 2-fold increase in receptor expression led to a ≥10-fold decrease in the concentration of ligand required to induce a biologic response (26). These findings suggest that autocrine TGFβ activity might result from receptor up-regulation rather than from an increase of ligand. Recently, we have demonstrated that the blockade of endogenous TGFβ signaling using anti-TGFβ antibody or TGFβ antisense oligonucleotide diminished the increased collagen production in SSc fibroblasts (13). Taken together, these results suggest that TGFβ signaling may play a central role in fibrotic disorder, and that the mechanism of regulation of TGFβRI and TGFβRII in such diseases is significant.

In conclusion, we have demonstrated that increased levels of TGFβRs in SSc fibroblasts play a critical role in excessive collagen production, and that up-regulation of TGFβR expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGFβR expression in SSc fibroblasts.

Acknowledgements

We thank Dr. R. H. Goldstein for kindly providing TGFβRI promoter luciferase constructs. We thank Dr. S.-J. Kim for kindly providing TGFβRII promoter luciferase constructs.

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