Epidermal growth factor up-regulates expression of transforming growth factor beta receptor type II in human dermal fibroblasts by phosphoinositide 3-kinase/Akt signaling pathway: Resistance to epidermal growth factor stimulation in scleroderma fibroblasts.
OBJECTIVE
Transforming growth factor beta receptors (TGFbetaRs) 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 TGFbetaR expression.


METHODS
The levels of expression of TGFbetaR type II (TGFbetaRII) 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 TGFbetaRII gene was examined with luciferase assays using the -1670/+35 TGFbetaRII promoter luciferase construct.


RESULTS
EGF up-regulates the expression of TGFbetaRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFbetaRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFbetaRII mRNA half-life. EGF-mediated induction of TGFbetaRII 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 TGFbetaRII. In addition, EGF induced TGFbetaRII 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 TGFbetaRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFbetaRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFbetaRII but did not show further up-regulation of TGFbetaRII expression by EGF.


CONCLUSIONS
These results indicate that EGF-mediated induction of TGFbetaRII 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 TGFbetaRII in scleroderma fibroblasts.
Yamane
Kenichi
K
Department of Dermatology, University of Tokyo, Tokyo, Japan. IN-DER@h.u-tokyo.ac.jp
Ihn
Hironobu
H
Tamaki
Kunihiko
K
D000730:Androstadienes / Q000494:pharmacology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D020405:Dermis / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D015854:Up-Regulation; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D003609:Dactinomycin; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D009025:Morpholines; D019384:Nucleic Acid Synthesis Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human