Gene Regulation ofCYP4F11in Human Keratinocyte HaCaT Cells

Abstract

ABSTRACT:Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans-and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor ␣ Cytochrome P450 4F enzymes are involved in cellular protection and metabolism of numerous small molecules, including drugs, toxins, and eicosanoids. To date, studies have been focused on CYP4F functions rather than transcriptional regulatory mechanisms. This approach is partially because of the apparent complexity of CYP4F gene regulation and differences observed among the various model systems studied.Despite the seeming complexity of CYP4F regulation, some research groups have made substantive contributions to this puzzle. Zhang and colleagues showed that retinoic acids and peroxisome proliferators can regulate CYP4F2 gene activities in HepG2 cells, and that retinoid X receptor (RXR) ␣ heterodimers stimulated and retinoic acid receptor (RAR) ␣ repressed CYP4F2 expression Hsu et al., 2007) showed that statins induced CYP4F2 in primary human hepatocytes and HepG2 cells. We recently showed that retinoic acids induced the expression of CYP4F2, CYP4F3A, CYP4F3B, and CYP4F11 in primary human epidermal keratinocytes, and that RXR (but not RAR) nuclear receptors mediate retinoic acid-induced up-regulation of CYP4F2 and CYP4F3A (Kalsotra et al., 2008). In a preliminary study (data not shown) using a transformed immortal human keratinocyte cell line (HaCaT), a different story unfolded. Lovastatin did not affect HaCaT cell CYP4F2 expression, which is in contrast to its up-regulatory effects on CYP4F2 in human hepatic cells (Hsu et al., 2007). In addition, 9-cis-retinoic acid failed to induce CYP4F11 in HaCaT cells; rather, it significantly decreased CYP4F11 transcript levels.Retinoic acids are derived from retinol, the animal form of vitamin A. Retinoic acids play a role in gene regulation via two groups of nuclear receptors (NRs), RARs and RXRs (Mangelsdorf et al., 1995

Regulation of Cytochrome P450 4F11 by Nuclear Transcription Factor-κB

“…We have shown that activation of the NF-B pathway by TNF-␣ stimulation or overexpression of MEKK causes down-regulation of CYP4F11 promoter transcripts. However, what makes this finding unique is that the inhibition of endogenous CYP4F11 is not seen in the presence of TNF-␣ after 24 h. This regulation by TNF-␣ was reported in a previous study from our laboratory (Wang et al, 2010) and is verified in this study in Fig. 1, which also shows that the presence of TNF-␣ and an inhibitor of NF-B cause a greater increase in CYP4F11 transcript quantity than TNF-␣ alone.…”

“…It has been shown that the JNK and the NF-B pathways are both stimulated in the presence of TNF-␣; however, they are competing forces (Papa et al, 2006). The up-regulation of CYP4F11 mRNA in the presence of TNF-␣ is due to the stimulation of the JNK pathway (Wang et al, 2010); however, this is time-dependent and does not reach a level of significance until 24 h after treatment, as observed in our experiments. This was confirmed by ChIP analysis, where there was a large increase in p65-bound CYP4F11 transcripts at 1 h after treatment that was not present at 24 h.…”

“…1, and also seen in the CYP4F11 promoter mutants after inhibition, can be explained partly by the activation of areas in the promoter region that have AP-1 binding sites, which would create higher levels of expression of CYP4F11 through JNK activation. This mechanism was described in an article published by our laboratory (Wang et al, 2010). In short, JNK activation causes activation of AP-1, which regulates expression through the AP-1 binding sites on the promoter region of the gene.…”

“…Previously published work reported that CYP4F11 expression is increased upon stimulation with TNF-␣ in keratinocyte HaCaT cells dmd.aspetjournals.org (Wang et al, 2010). TNF-␣ activates two pathways: the JNK pathway, which was shown to up-regulate CYP4F11 expression through activator protein 1 (AP-1) binding sites, and the NF-B signaling pathway.…”

“…TNF-␣ stimulation causes an activation of two signal transduction pathways, the c-Jun NH 2 -terminal kinase (JNK) pathway and the nuclear factor B of the light-chain-enhancer in activated B cells (NF-B) pathway (De Smaele et al, 2001;Tang et al, 2001;Deng et al, 2003). Our previous studies have examined the roles the JNK pathway and retinoic acids play in the regulation of CYP4F11 and have found that JNK stimulation causes an increase in CYP4F11 mRNA, whereas retinoids cause down-regulation of CYP4F11 mRNA (Wang et al, 2010). However, we did not examine the effects of NF-B on CYP4F11 expression during TNF-␣ stimulation.…”