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.
Interleukin-6 (IL-6) is produced during bacterial and viral infections and by various malignant tumors. Here, we describe novel immunosuppressive properties of IL-6 in dendritic cells (DC). In the presence of GM-CSF, IL-4, and a maturation stimulus, IL-6 skewed monocyte differentiation into phenotypically mature but functionally impaired DC. In DC matured with the toll-like receptor (TLR)4 stimulus lipopolysaccharide (LPS) or other pro-inflammatory stimuli, IL-6 inhibited CCR7 chemokine receptor up-regulation. As demonstrated for LPS-stimulated DC, IL-6 impaired chemotaxis to CCR7-activating chemokines required for recruiting DC to lymphoid tissues in vivo. Moreover, IL-6 inhibited production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma inducible protein-10 (IP-10) in DC, and DC-driven allogeneic T cell proliferation in mixed lymphocyte reactions. CCR7 expression was blocked at the transcriptional level. IL-6 led to inhibition of nuclear factor-kappaB (NF-kappaB) binding activity, regulating CCR7 transcription. Neutralization experiments revealed that autocrine IL-10 partially contributed to CCR7 suppression in IL-6-treated DC. Thus IL-6, a cytokine once labeled as "pro-inflammatory" can mediate immunosuppressive functions, which may involve induction of the classical "anti-inflammatory" cytokine IL-10. Because IL-6 is expressed in response to various pro-inflammatory stimuli in vivo, this mechanism may contribute to down-regulating the immune response initiated by pathogens, in persistent infections or tumors.
Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue. They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGFbeta activity and also induces fibroblast to differentiate into alpha-smooth muscle actin (alphaSMA)-positive myofibroblasts. TGF-beta activity alone cannot completely account for alphaSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally alphaSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of alphaSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce alphaSMA expression; however, ET-1 did synergize with TGF-beta. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited alphaSMA expression in fibroblasts, costimulated with TGF-beta and ET-1. GM-CSF activated NFkappaB, thus interfering with TGF-beta signaling. Blocking TGFbeta and ET-1 largely impaired alphaSMA induction in co-cultures at day 7 and, in combination, almost completely prevented alphaSMA induction. Our results dissect the roles of TGF-beta and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.
The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBP␣ and - in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBP sites and the C/EBP␣-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBP␣ or - coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBP. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions.Human papillomaviruses (HPVs) are small, double-stranded DNA viruses causing proliferative lesions of the skin or mucosa. The viral life cycle is closely linked to the keratinocyte's differentiation program. According to the current model, HPVs infect keratinocytes of the basal cell layer via microtraumata. In these cells the early viral genes start to be weakly expressed and a maintenance copy number of the viral genome is established. Normally, keratinocytes have to exit the cell cycle in order to differentiate. As HPVs depend on the host cell's DNA-dependent DNA polymerase, the viral oncoproteins E6 and E7 maintain keratinocytes in a proliferative stage, thus enabling vegetative viral DNA replication in the upper layers of the epithelium. Furthermore, E6 and E7 inhibit terminal keratinocyte differentiation induced by serum or calcium (39, 49). The early protein E1 and the viral transcription factor E2 play a major role in the initiation of viral replication. All E2 proteins share a common modular structure containing an N-terminal transactivation domain and a C-terminal DNA binding and dimerization domain. These highly conserved domains are linked by the less conserved hinge region. The E2 protein of HPV type 16 (HPV16) was found to be expressed in the superficial layers of cervical intraepithelial neoplasia I (CINI) and CINII lesions and only at low levels in the intermediate and basal layers. In CINIII lesions, E2 is only weakly expressed, and in squamous cell carcinomas it is absent (44). In a benign cutaneous lesion of a patient suffering from epidermodysplasia verruciformis, E2-specific mRNA was detected in the upper two-thirds of the epidermis (15). E2 not only regulates E6 and ...
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