Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-β and Interleukin-1

Abstract

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.

Squamous metaplasia amplifies pathologic epithelial-mesenchymal interactions in COPD patients

“…In contrast, in a study utilizing a coculture model of keratinocytes and dermal fibroblasts, myofibroblast differentiation was TGF-β dependent but was antagonized by IL-1β (52). The discrepancy between these 2 studies likely represents inherent cell type-specific differences between airway and dermal fibroblasts.…”

“…*P < 0.05; **P < 0.001. link innate immune responses to subsequent adaptive immune responses, ultimately promoting wound repair (50). Thus, the induction of IL-1β that occurs during SM of the airway epithelium may act as both an autocrine factor to influence gene expression to facilitate cell migration and wound closure and as a paracrine factor to recruit inflammatory cells and to induce matrix production and contractility of adjacent fibroblasts (51,52). The basal, suprabasal, and superficial cells of SM in COPD all express increased IL-1β, suggesting that both increased luminal and basal secretion of IL-1β are provided by the squamous metaplastic epithelium.…”

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

“…This is the first study showing the inhibition of CMF differentiation by IL-1α. Earlier studies using co-cultures of keratinocytes and dermal fibroblasts showed that TGFβ1-induced αSMA expression was suppressed by IL-1α (Shephard et al, 2004). Moreover, blocking the IL-1 receptor potentiated αSMA expression in the dermal fibroblasts, indicating an inhibitory effect of IL-1 on TGFβ1-induced myofibroblast differentiation (Shephard et al, 2004).…”

“…Earlier studies using co-cultures of keratinocytes and dermal fibroblasts showed that TGFβ1-induced αSMA expression was suppressed by IL-1α (Shephard et al, 2004). Moreover, blocking the IL-1 receptor potentiated αSMA expression in the dermal fibroblasts, indicating an inhibitory effect of IL-1 on TGFβ1-induced myofibroblast differentiation (Shephard et al, 2004). In human pulmonary fibroblasts, TGFβ1-induced αSMA expression was inhibited by TNFα, indicating that other pro-inflammatory cytokines show similar inhibitory effects on myofibroblast differentiation (Liu et al, 2009).…”

Evaluation of Myofibroblasts By Expression of Alpha Smooth Muscle Actin: A Marker in Fibrosis, Dysplasia and Carcinoma

“…Cytoskeleton proteins, ECM molecules, Cytokines and Nucleus proteins which are elements of Epithelial Mesenchymal Transition (EMT), which are commonly associated with OSMF, lead to the possibility of EMT being a part of fibrosis of oral mucous, which is triggered by betel quid chewing habit in OSMF [6]. Extensive myofibroblast differentiation requires close contact between keratinocytes and fibroblasts, which suggests their role in cellular signaling in dermal wounds [7].…”