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

Abstract

Objective. Transforming growth factor ␤ receptors (TGF␤Rs) 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 TGF␤R expression.Methods. The levels of expression of TGF␤R type II (TGF␤RII) 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 TGF␤RII gene was examined with luciferase assays using the ؊1670/؉35 TGF␤RII promoter luciferase construct.Results. EGF up-regulates the expression of TGF␤RII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGF␤RII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this upregulation. In addition, EGF treatment did not significantly affect the TGF␤RII mRNA half-life. EGFmediated induction of TGF␤RII 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 TGF␤RII. In addition, EGF induced TGF␤RII 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 TGF␤RII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGF␤RII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGF␤RII but did not show further up-regulation of TGF␤RII expression by EGF.Conclusion. These results indicate that EGFmediated induction of TGF␤RII 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 EGFmediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGF␤RII in scleroderma fibroblasts.Transforming growth factor ␤ (TGF␤) is a multifunctional protein that plays an important role in regulating cellular growth, differentiation, adhesion, and apoptosis in many biologic systems (1-3). TGF␤ inhibits the growth of most cell types. In addition, TGF␤ 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-

Autocrine TGF-β signaling in the pathogenesis of systemic sclerosis

“…Increased expression of TGF-b receptors type I and type II has also been reported in SSc fibroblasts in vitro and in vivo [14,[117][118][119][120]. The overexpression of these TGF-b receptors in SSc fibroblasts was shown to be regulated at transcriptional level [118] and to be resistant to various stimuli [119,120].…”

“…The overexpression of these TGF-b receptors in SSc fibroblasts was shown to be regulated at transcriptional level [118] and to be resistant to various stimuli [119,120]. TGFb receptors have been shown to be up-regulated by TGF-b, PDGF and EGF [119,121,122]. Moreover, elevated expression of the endothelial cell-enriched TGF-b receptor endoglin was reported in SSc fibroblasts [123].…”

Novel Permissive Role of Epidermal Growth Factor in Transforming Growth Factor β (TGF-β) Signaling and Growth Suppression

“…However, unlike in LNCaP cells, in NRK-49F cells EGF only partially reverses the loss of T␤RII expression by TGF-␤1. NRK-49F cells may behave similarly to human dermal fibroblasts, in which EGF was shown to activate the T␤RII promoter through a PI3-kinase-dependent mechanism (68).…”

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

“…Phosphorylated levels of Smad3 or p85 were examined by immunoprecipitation using anti-Smad3 and antiphosphoserine-specific antibodies or anti-p85 and anti-phosphotyrosine antibodies, respectively (Ihn et al, 2001a;Yamane et al, 2003b). The same membrane was then stripped and reprobed with anti-Smad2/3 (N-19) or p85 antibody to show the total amount of Smad3 or p85, respectively.…”