Epidermal growth factor up‐regulates expression of transforming growth factor β receptor type II in human dermal fibroblasts by phosphoinositide 3‐kinase/Akt signaling pathway: Resistance to epidermal growth factor stimulation in scleroderma fibroblasts

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Abstract
Objective 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. Methods 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. Results 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. Conclusion 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.