Endothelin 1 contributes to the effect of transforming growth factor beta1 on wound repair and skin fibrosis.
OBJECTIVE
To characterize the pathways induced by transforming growth factor beta1 (TGFbeta1) that lead to the expression of endothelin 1 (ET-1) in human dermal fibroblasts, and to study the effects of TGFbeta1 and ET-1 on the acquisition of a profibrotic phenotype and assess the contribution of the TGFbeta1/ET-1 axis to skin wound healing and fibrosis in vivo.


METHODS
The mechanism of induction of ET-1 expression by TGFbeta1 and its effect on the expression of alpha-smooth muscle actin and type I collagen were studied in human dermal fibroblasts, in experiments involving the TGFbeta receptor inhibitor GW788388 and the ET receptor antagonist bosentan, by real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, immunofluorescence, Western blotting, and promoter/reporter transient transfection analyses. Experiments assessing dermal wound healing in mice were performed with adenovirus-driven overexpression of active TGFbeta1 and ET-1, with or without treatment with bosentan. The contributions of TGFbeta1 and ET-1 to the fibrotic response were also assessed in a mouse model of bleomycin-induced skin fibrosis, by histologic, immunohistochemical, RT-PCR, and protein analyses.


RESULTS
TGFbeta1 induced ET-1 expression in human dermal fibroblasts through Smad- and activator protein 1/JNK-dependent signaling. The ability of TGFbeta1 to induce the expression of profibrotic genes was dependent on ET-1. Adenovirus-mediated overexpression of TGFbeta1 and ET-1 in mouse skin was associated with accelerated wound closure, increased fibrogenesis, and excessive scarring. Treatment with bosentan prevented the effects of TGFbeta1. In the bleomycin-induced fibrosis model, treatment with GW788388 and bosentan prevented the fibrotic response.


CONCLUSIONS
Our results strongly support the notion that the TGFbeta1/ET-1 axis has a role in wound repair and skin fibrosis. ET-1 receptor antagonists, such as bosentan, may represent a useful therapeutic tool in the treatment of excessive scarring and fibrosis-related diseases.
Lagares
David
D
Laboratorio Mixto Fundación Renal Iñigo Alvárez de Toledo, FRIAT-CSIC, Madrid, Spain.
García-Fernández
Rosa Ana
RA
Jiménez
Clara López
CL
Magán-Marchal
Noemi
N
Busnadiego
Oscar
O
Lamas
Santiago
S
Rodríguez-Pascual
Fernando
F
D000199:Actins / Q000032:analysis; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001761:Bleomycin; D015153:Blotting, Western; D000077300:Bosentan; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis; D019332:Endothelin-1 / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D005355:Fibrosis / Q000503:physiopathology; D051379:Mice; D008809:Mice, Inbred C3H; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000187:drug effects / Q000473:pathology*; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology*
C509927:4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide; D000199:Actins; D001549:Benzamides; D024042:Collagen Type I; D019332:Endothelin-1; D011720:Pyrazoles; D012333:RNA, Messenger; D013449:Sulfonamides; D053773:Transforming Growth Factor beta1; D001761:Bleomycin; D000077300:Bosentan