In addition to NOS, arginine is a substrate for arginase. In the present study, we detected the upregulation of arginase II mRNA in our in vitro GA model (
Fig. 4). Arginase II upregulation contributes to several diseases, such as vasoregulatory systemic dysfunction,
25,26 pulmonary artery hypertension,
27 diabetic-associated erectile dysfunction,
28 and atherosclerosis.
29 Previous studies have also shown that arginase II is transcriptionally regulated by lipopolysaccharide in macrophages,
30,31 liver X receptors, which have been implicated in lipid metabolism and inflammation in macrophages,
32 interferon regulatory factor 3 (IRF3) in T lymphocytes,
33 and cAMP in Caco-2 tumor cells.
34 In the present experiments, the upregulation of arginase II in OAT-deficient hTERT-RPE cells was triggered by ornithine but not by ornithine metabolites with ODC. We found that ornithine increased arginase II expression in a dose-dependent manner (
Fig. 5A), whereas DFMO and ODC siRNA did not reduce the induction of arginase II, but rather enhanced it (
Figs. 5B,
5D). The enhancement of arginase II might have been due to the maintenance of ornithine concentration entailing ODC deficiency. We also noted that the maximal induction of CAT-1 preceded that of arginase II (
Fig. 4B). Furthermore, the addition of 5-FMO enhanced the expression of arginase II mRNA induced by ornithine (
Fig, 5A), suggesting that the accumulation of ornithine by OAT deficiency is the result of arginase II upregulation. On the other hand, the NOS inhibitor L-NAME partially inhibited the upregulation of arginase II induced by ornithine in OAT-deficient hTERT-RPE cells (
Fig. 6A), suggesting that NO takes part in the induction of arginase II in addition to ornithine. This was further supported by our findings showing that arginase II upregulation was detected in cells treated with arginine (
Fig. 5E), that the induction by arginine was unaffected by an arginase inhibitor BEC (
Fig. 5F), and the NO donor SNAP increased the level of arginase II mRNA (
Fig. 6B). Similarly, CAT-1 expression was upregulated by SNAP in hTERT-RPE cells, though CAT-1 mRNA has also been shown to be upregulated by polyamines, which are ornithine metabolites.
12 Because NO has demonstrated abilities to both upregulate and downregulate the expression of genes in mammalian cells,
35,36 another possible mechanism of NO for cytotoxicity in hTERT-RPE cells appears to be the transcriptional regulation of arginase II and CAT-1. Ornithine transiently upregulated iNOS expression after 1 hour in OAT-deficient hTERT-RPE cells (
Fig. 2B), whereas the upregulation of arginase II and CAT-1 started at 3 and 6 hours, respectively, after treatment (
Fig. 4B). The initial induction of iNOS may be involved in the deterioration associated with progressive cytotoxicity of RPE. There is no report showing the induction of arginase II or iNOS by treatment with ornithine, though the induction of CAT-1 by polyamine is involved in transcription factor c-myc.
12 Some transcription factors, such as liver X receptor,
32 IRF3,
33 and cAMP-response element binding protein (CREB),
34 have been suggested to induce arginase II based on the use of chemical inhibitors. The induction of iNOS expression is regulated by many transcription factors. cAMP enhances iNOS induction in some cell types, including rat RPE-J cells
20 ; therefore, CREB might be used as a common transcription factor for arginase II and iNOS induction.