Myofibroblast differentiation is induced in keratinocyte-fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1.
In wound healing epidermal-dermal interactions are known to regulate keratinocyte proliferation and differentiation. To find out how fibroblasts respond to epithelial stimuli, we characterized fibroblasts in monolayer co-culture with keratinocytes. On co-culture numerous extracellular matrix- and smooth muscle cell-associated gene transcripts were up-regulated in fibroblasts, suggesting a differentiation into myofibroblasts. Increased alpha-smooth muscle actin (alpha-SMA) protein expression in co-cultured fibroblasts started at approximately day 4, was serum-independent, but required endogenous transforming growth factor (TGF)-beta. In co-cultures, TGF-beta neutralizing monoclonal antibody strongly reduced alpha-SMA induction. Endogenous TGF-beta production and activation were increased at 24 and 48 hours, requiring, like alpha-SMA induction, close keratinocyte-fibroblast proximity. As myofibroblast differentiation only started after 4 days, we analyzed the presence of endogenous inhibitors at early time points. Blocking keratinocyte-derived interleukin (IL)-1 using IL-1 receptor antagonist, alpha-SMA expression in co-cultures was potentiated. Conversely, adding exogenous IL-1alpha completely suppressed endogenous alpha-SMA induction. In co-cultured fibroblasts strong nuclear factor-kappaB binding activity was observed from 2 hours, decreasing at 2 and 4 days, suggesting an early, IL-1-mediated inhibition of TGF-beta signaling in co-cultured fibroblasts. This biphasic differentiation event is regulated by the balance of endogenous TGF-beta and IL-1 activity and is reminiscent of myofibroblast differentiation at early and later stages of wound healing.
Shephard
Pierre
P
Departments of Dermatology and Virology, University of Cologne, Cologne, Germany.
Martin
Gail
G
Smola-Hess
Sigrun
S
Brunner
Georg
G
Krieg
Thomas
T
Smola
Hans
H
D000328:Adult; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D005347:Fibroblasts / Q000166:cytology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D015603:Keratinocytes / Q000166:cytology*; D032342:Muscle Cells / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D012867:Skin / Q000166:cytology; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D007375:Interleukin-1; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta