The Expression And Function Of F1F0-ATPase(Synthase)in Keratinocyte Differentiation

ObjectiveF1F0-ATP ase (synthase) is a widely expressed protein complex. It is located on the inner membrane of mitochondria or sometimes cell membrane to generate or hydrolyze ATP, which makes it an important part of cell OXPHOS system. In this study, we focused on the expression of FIFO-ATP ase (synthase) during the differentiation of kerationcyte and the possible roles it may play, based on which, we hope to give some new insights into the relationship between cell metabolism, intracellular and extracellular ATP level and keratinocyte differentiation.Methods1. The expression of ATP5B in normal skin, psoriasis, squamous cell carcinoma and keratoacanthoma, were detected by immunohistochemistry.2. Based on the cell model we established of the indensity-induced (confluency-induced) HaCaT differentiation, the expression of the β subunit of FIFO-ATP ase (synthase)(ATP5B) were tested by Western Blot and RT-PCR, respectively.3. The impact of small-molecule inhibitors (oligomycin and piceatannol) on the intracellular and extracellular ATP were detected by ATP quantitative bioluminescence assay, and the impact on the expression of differentiation biomarker (involucrin) were detected by Western Blot.Results1. The expresstion of ATP5B in normal skin, psoriasis and keratoacanthoma, increases paralled with epidermis differentiation, with visibly higher expression in normal skin than in other diseases.2. We established the cell model of indensity-induced HaCaT differentiation, the expression of differentiation biomarkers was confirmed at the mRNA and protein level, respectively.3. We observed a marked increase of ATP5B at mRNA level at6d and8d of the differentiation cell model, however with little change at protein level.4. Under normal cell culture condition, oligomycin can markedly reduce intracellular ATP and the expression of involucrin; piceatannol mainly reduces extracellular ATP rather than intracellular ATP, and it did not reduce involucrin expression.5. In the differentiation cell model, piceatannol reduced the intracellular ATP level and involucrin expression at the later stage of the differentiation cell model.Conclusion1. The expression of F1F0-ATPase(synthase) may increase parrelled with keratinocyte differentiation and it is reduced in psoriasis, squamous carcinoma and some other common abnormal-epidermis-proliferation diseases.2. The inhibitors of F1F0-ATPase (synthase) may block the differentiation of HaCaT cell line, which may be related with the decrease of intracellular ATP.