Differential transcriptional responses of keloid and normal keratinocytes to serum stimulation.
BACKGROUND
Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs.


METHODS
Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively.


RESULTS
Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs.


CONCLUSIONS
KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.
Department of Surgery, Stanford University Medical School, Stanford, California, USA.
Wei
W
Xia
Toan-Thang
TT
Phan
Ivor J
IJ
Lim
Michael T
MT
Longaker
George P
GP
Yang
D000293:Adolescent; D000328:Adult; D000873:Anthracenes / Q000494:pharmacology; D001798:Blood Proteins / Q000494:pharmacology*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007627:Keloid / Q000473:pathology* / Q000503:physiopathology*; D015603:Keratinocytes / Q000187:drug effects* / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000873:Anthracenes; D001798:Blood Proteins; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D011725:Pyridines; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; C432165:pyrazolanthrone; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one