The Effect Of Disorder Of Mevalonate Pathway On The Proliferation, Differentiation And Apoptosis Of Primary Kerationcytes

Objective To explore the effect of disorder of mevalonate pathway on proliferation, differentiation and apoptosis of primary kerationcytes and its possible molecμlar mechanism and to provide the basis for clearly understanding the pathologic and molecular mechanism of disseminated superficial actinic porokeratosis (DSAP).Methods (1) After KCs were cultured, specific inhibitors of key enzyme which catalyze the smoothy proceeds of mevalonate pathway, such as HMGCoA reductase inhibitor Pravastatin (PRA), Farnesyl synthetase inhibitor (ALD), Farnesyl transferase inhibitor (FTI-277) and Geranylgeranyl transferase inhibitor (GGTI-298) were used alone or combination with some intermediates (mevalonic acid (MVA), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP),cholesterol(CH)) of mevalonate pathway for 2 days. MTS assay and flow cytometry were used to analyze its effect on cell viability and cell cycle. After extracting the total protein, western-blot was used to detect the expression of cycle-associated protein, including cyclinB1, cyclinE and p21 and analyze its effect on MAPK-ERK1/2 and PI3K-AKT. (2) After treating like the steps above for 2 days,1.2mM Ca2+ were added to co-culture forl day, then total protein were extracted. Western-blot was used to detect the differentiatin makers (Involucrin, Keratin1) induced by Ca2+ and analyze the p53, Notchl and MAPK-ERK1/2, PI3K-AKT. (3) After KCs were treated like the way above, flow cytometry was used to detect its effect on apoptosis of KCs.Results (1) Proliferation results:MTS results indicated that PRA and ALD strongly inhibited cell proliferation. FTI, GGTI also inhibited the cell proliferation, but the effect was weaker. Only CH could rescue the effect of proliferation inhibition caused by PRA or ALD. MVA, FPP and GGPP induced partial inhibition effect. Flow cytometry results of cell-cycle indicated that PRA and ALD cause G1 arrest. Meanwhile, CH could rescue the effect of cycle arrest caused by both PRA and ALD. FPP, GGPP could also caused Glarrest.Moreover, FTI, GGTI didn’t influence the cell cycle. Western-blot analysis suggested that PRA, ALD, FTI, GGTI alone or jointly down-regulate the expression of cyclinB1, while up-regualte expression of cyclinE, p21; and they also activated the PI3K-AKT signal pathway and inhibited activation of MAPK-ERK1/2. (2) Differentiation results:Western-blot analysis showed that PRA, ALD, FTI, GGTI alone or jointly inhibited the expression of Involucrin. All the inhibitors alone or jointly could down-regulate expression of p53, Notch1. The results of signal pathway indicated that Ca2+ activated the AKT while inhibited the ERK1/2. ALD alone or four inhibitors up-regulated the phosphorylation of AKT, while PRA, FTI, GGTI alone all decreased level of the phosphorylation of AKT and increased the phosphorylation of ERK1/2. (3) Flow cytometry results of apoptosis showed that all of inhibitons or products didn’t induce the apoptosis of KCs.Conclusion Disorder of mevalonate mathway inhibited proliferation, differentiation, but didn’t influence the apoptosis of KCs.