Abstract
Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.
Highlights
Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7
The addition of EGF, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-␣ increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of autoand cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-␣
We demonstrate that the mRNA expression profile upon induction of differentiation reveals the differential expression of epiregulin, HB-EGF, AR, and TGF-␣ in keratinocytes
Summary
Materials—Recombinant EGF and TGF-␣ were generous gifts from Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). The following day, the cells were re-fed again with the same medium containing various concentrations of recombinant epiregulin and incubated for 24 h. Incorporation of BrdUrd was determined using a cell proliferating enzyme-linked immunosorbent assay system (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. 5 ml of MCDB153 containing 0.5 mCi of [35S]methionine (Amersham Pharmacia Biotech) was added to the cells, and they were cultured for 2 days. Samples were solubilized in 0.5 ml of PBS(Ϫ) and quantified by an enzyme-linked immunosorbent assay kit (Otsuka Pharmaceutical Co., Ltd., Tokyo) according to the instructions of the manufacturer. 20 g of total RNA/lane was loaded and separated by electrophoresis on a 1.2% formaldehyde-agarose gel and transferred to a nylon membrane (Hybond Nϩ, Amersham Pharmacia Biotech) in 20ϫ SSC (150 mM sodium chloride, 15 mM sodium citrate) according to standard procedure. A cycle profile consisted of 1 min at 94 °C for denaturation, 1.5 min at 60 °C for annealing, and primer extension. 5 l of each reaction mixture was electrophoresed on a 2.0% agarose gel containing ethidium bromide to evaluate amplification
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