Impact of aryl hydrocarbon receptor (AhR) knockdown on cell cycle progression in human HaCaT keratinocytes.

Abstract While activation of the aryl hydrocarbon receptor (AhR) by exogenous ligands is well investigated, its physiological function is less understood. By extending research in AhR biology, evidence appeared that the receptor generally plays an important role in cell physiology. In keratinocytes, little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on cell cycle progression in HaCaT cells and showed that proliferation of siAhR HaCaT cells was significantly decreased. In line with that result, western blot analysis revealed that protein level of the cyclin dependent kinase inhibitor p27(KIP1) was increased, whereas protein level of the cyclin dependent kinase (CDK) 2 was reduced. CDK4 and CDK6 protein levels remained unchanged, whereas protein level of the retinoblastoma protein (pRB) was reduced. By measuring ethoxyresorufin-O-deethylase (EROD) activity we showed that endogenous cytochrome P450 1 (CYP1), especially CYP1A1 is required for normal cell cycle in HaCaT cells, as well. To the best of our knowledge, we provide evidence for the first time in human skin cells, that in the absence of exogenous ligands, the AhR promotes cell cycle progression in HaCaT cells and one can speculate that this is the physiological function of this receptor in keratinocytes.

Characterization of N-acetyltransferase 1 activity in human keratinocytes and modulation by para-phenylenediamine.

N-acetyltransferase 1 (NAT1)-mediated N-acetylation in keratinocytes is an important detoxification pathway for the hair dye ingredient para-phenylenediamine (PPD). Because NAT1 can be regulated by various exogenous compounds, including some NAT1 substrates themselves, we investigated NAT1 expression in keratinocytes and the interactions between PPD and NAT1. NAT1 activity was found to be cell-cycle phase-dependent. Maximum NAT1 activities (mean: 49.7 nmol/mg/min) were estimated when HaCaT keratinocytes were arrested in G(0)/G(1) phase, whereas nonsynchronized cells showed the lowest activities (mean: 28.9 nmol/mg/min). It is noteworthy that we also found an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. This evidence suggests an association between NAT1 and proliferation in keratinocytes. Regarding the interaction between NAT1 and PPD, we found that keratinocytes N-acetylate PPD; however, this N-acetylation was saturated with increasing PPD concentrations. HaCaT cultured in medium supplemented with PPD (10-200 microM) for 24 h showed a significant concentration-dependent decrease (17-50%) in NAT1 activity. PPD also induced down-regulation of NAT1 activity in human primary keratinocytes. Western blot studies using a NAT1-specific antibody in HaCaT showed that the loss of enzyme activity was associated with a decline in the amount of NAT1 protein, whereas no changes in the amounts of NAT1 P1 (NATb)-dependent mRNA were found by quantitative reverse transcription-polymerase chain reaction analysis, suggesting the involvement of a substrate-dependent mechanism of NAT1 down-regulation. In conclusion, these data show that overall N-acetylation capacity of keratinocytes and consequently detoxification capacities of human skin is modulated by the presence of NAT1 substrates and endogenously by the cell proliferation status of keratinocytes.