In this report, we found that p38 kinase regulates VES-induced NAG-1 mRNA/protein expression via transcriptional/post-transcriptional mechanism and NAG-1 expression is important in VES-induced apoptosis.
Yu et al. (47
) reported that the activation of ERK and JNK but not p38 is required for VES-induced apoptosis of human breast cancer cells. We observed the increased level of phospho-JNK after VES treatment (); however, pretreatment with a JNK inhibitor did not block VES-induced NAG-1 () and cell death (data not shown). This discrepancy may be due to different time points (12 versus 48 h), different VES concentration (20 μg/mL = 37 versus 20 μmol/L), or different cell type (MDA-MB-435 human breast cancer cells versus PC-3 human prostate carcinoma cells). In addition, we have determined if an inhibition of protein kinase A affect VES-induced NAG-1 expression using protein kinase A inhibitor (H-89). However, an inhibition of protein kinase A did not affect VES-induced NAG-1 expression, although H-89 treatment blocked phosphorylation of CREB (data not shown).
Because VES treatment induced NAG-1 promoter reporter activity, we tried to determine the transcription factor that activates NAG-1 promoter in response to VES treatment. NAG-1 has been shown to be a target gene for several transcription factors, such as p53, Egr-1, ATF-3, and Sp1. In addition, p38 kinase has been shown to phosphorylate and activate several transcription factors, such as MEF2, ATF-2, CHOP, Ets-1, Elk-1, and p53. We have examined if VES treatment activates or induces expression of above-mentioned transcription factors in a p38 kinase-dependent manner. Based on gel shift, Western blot analysis, or NAG-1 promoter reporter assays, none of the known p38 target transcription factors were responsible for VES-induced NAG-1 expression (data not shown). Furthermore, the transcriptional activity or the expression level of Egr-1 or Sp1 was not changed by VES treatment. Interestingly, the level of ATF-3 expression was significantly increased by VES treatment. However, the increase in ATF-3 expression by VES was p38 kinase independent but protein kinase A dependent (data not shown), indicating that ATF-3 is not responsible for VES-induced NAG-1 expression. These results suggest that VES may activate or induce expression of novel p38 target transcription factor.
Although NAG-1 expression is induced by many stimuli via transcriptional activation, some chemopreventive/chemotherapeutic chemicals, such as MCC-555 (46
) and 5F-203 (48
), have been shown to increase NAG-1 mRNA levels by stabilizing NAG-1 mRNA levels. The regulation of mRNA stability is the important mechanism to control certain gene expression where the stability of mRNA is determined by interactions between specific sequences within mRNA (cis
-acting elements) and cellular RNA-binding proteins (trans
-acting factors). One of the best characterized cis
-acting elements is the ARE which exists in the 3′-UTR of mRNAs of cytokines or growth factors and NAG-1 has four well-conserved copies of ARE (46
). Because VES treatment activates p38 kinase and p38 kinase regulates the stability of certain mRNAs, such as tumor necrosis factor-α and Cox-2, we hypothesized that VES may stabilize NAG-1 mRNA and found that VES stabilize NAG-1 mRNA. ARE regulates the stability of mRNAs via the interaction with RNA-binding proteins. There are several known ARE-binding proteins, such as HuR, AUF1, TIA-1, and TTP, which bind to ARE and stabilize/destabilize mRNA. We examined the level of these mRNA-binding proteins after VES treatment. HuR is a member of RNA-binding proteins (49
) and known to bind/stabilize AREs in a p38 kinase-dependent manner. However, based on Western blot analysis, the level of HuR protein was not changed significantly and nuclear/cytoplasmic distribution of HuR protein was not changed by VES treatment (data not shown). The level of TTP mRNA, ARE-binding protein that destabilizes mRNA, was increased by VES treatment (data not shown), which is consistent with the finding that p38 pathway up-regulate TTP expression by stabilizing ARE in TTP mRNA (50
). However, the forced expression of TTP did not affect endogenous level of NAG-1 (data not shown). In addition, the level of AUF1 mRNA was decreased by VES treatment (data not shown). Because VES treatment affected the level of these ARE-binding proteins, it is possible that VES may regulate the expression of growth/apoptotic genes by changing their mRNA stability. Further studies will focus which ARE-binding proteins are involved in VES-induced stabilization of NAG-1 mRNA.
Our results indicate that VES-induced NAG-1 expression could contribute the antitumorigenic activity of VES. The level of NAG-1 expression was only significant at 10 and 20 μmol/L of VES treatment (), at which significant growth arrest and cell death occurs (), indicating the possible role of NAG-1 in VES-induced growth arrest or cell death. NAG-1-expressing PC-3 cells that stably express NAG-1 resulted in ~50% reduction in final tumor weight1
in nude mice xenograft experiment, which is consistent with previous findings (36
). Furthermore, the expression of NAG-1 siRNA significantly blocked VES-induced apoptosis (). However, VES seems to affect many other signaling molecules which are important for cell growth/survival because NAG-1 siRNA expression was not able to completely block VES-induced apoptosis. In summary, we have shown that VES, which is a chemopreventive compound in prostate cancer, induces NAG-1 expression in a p38 kinase-dependent mechanism and these findings suggest NAG-1 as a possible therapeutic target against the prostate cancer.