Transcriptional Regulation of the Human Neutral Ceramidase Gene

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Many forms of cellular stress cause an elevation of endogenous, apoptotic, ceramide (Cer) levels. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine‐1‐phosphate. To further define the physiological regulation of neutral CDase (nCDase), we used a luciferase reporter analysis system to define the proximal promoter of the nCDase gene and to demonstrate that serum can activate this proximal promoter, which corresponded to a serum‐induced increase in human nCDase mRNA expression. Computational analysis identified candidate transcriptional response elements (TRE), and electrophoretic mobility shift assays (EMSA) demonstrated that these TRE bind their respective transcription factors, including NF‐Y, AP‐2, Oct‐1, GATA, and serum‐activated AP‐1. Mutagenic analyses of the TRE revealed that these sites, including the AP‐1 cis‐element, regulate the promoter and are necessary for maximal activity of the proximal promoter. Moreover, siRNA knockdown of the AP‐1 subunit, c‐Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c‐Jun overexpression was sufficient to increase its activity. Thus, the transcription factor AP‐1 can directly regulate the nCDase promoter. Taken together, our findings suggest that c‐Jun/Ap‐1 signaling may regulate human nCDase gene transcription. These studies were funded by NIH HL076789 to MK.