Curcumin is a chemical component extracted from the rhizome of Zingiberaceae and Araceae. Studies have described various effects of curcumin including reducing blood fat, choleretic actions, and anti-tumor, anti-inflammatory, and anti-oxidation effects [13–16]. Several studies have indicated that the effect of curcumin on SREBPs is related to the mechanism of curcumin regulating blood lipid metabolism [4, 5, 8–12]. Kumar et al [4] found that curcumin can regulate the expression of NPC1L1 through SREBP-2 to inhibit cholesterol absorption in Caco-2 cells. We used a mouse model of cholesterol gallstone formation induced by a high-fat diet to show these results. Our preliminary study further confirmed that curcumin can reduce cholesterol gallstone formation and inhibit NPC1L1[5]. As we investigated how curcumin affects NPC1L1 expression through SREBP-2, we found different results from previous studies. Although curcumin lowered SREBP-2 mRNA levels, as in other studies, we did not see simultaneously down-regulated SREBP-2 protein levels. In the Caco-2 cell line, SREBP-2 mRNA expression decreased with higher doses (Fig. 1a) and times (Fig. 1b) of curcumin treatment. However, we did not find pSREBP-2 precursor levels to be inhibited (Fig. 2). Western blotting showed that while pSREBP-2 (almost 130 kDa) wasn’t significantly down-regulated, the amount of mSREBP-2 protein (55–70 kDa) decreased (Fig. 2). The SREBP-2 mRNA levels were inconsistent with pSREBP-2 levels, which was particularly evident through the curcumin time course. Regardless of whether curcumin was administered long-term (0 ~ 72 h) or short-term (within 24 h), the pSREBP-2 did not decrease following with the mRNA inhibition. Kang et al [8] found that the curcumin-mediated reduction of SREBP-2 promoter activity was dependent on the inhibition of SP-1. Consistent with Kang’s study, we also found downregulated transcriptional and translational levels of SP-1 at 24 h treatment, but this down-regulation was relieved after 48 h treatment (Fig. 1b, Fig. 2b). This suggests that curcumin’s effect on SREBP-2 may not involve SP-1, or that there may be another pathway. Considering the amount that mSREBP-2 protein decreased after curcumin treatment and that SREBP-2 is activated as a transcription factor through the proteolytic process [6, 7], we hypothesized that the effect of curcumin on SREBP-2 may depend on its proteolytic process. Endoplasmic reticulum co-localization showed that curcumin modulated SREBP-2 distribution within the cell, decreased mSREBP-2 in the nucleus, and inhibited the proteolysis process (Fig. 3c). In Brown and Goldstein's research [6, 7], they suggest that the SREBP-2/SCAP complex is the real substrate of S1P and key for the proteolytic process of SREBP-2. Therefore, we also detected SCAP in our study. SCAP and SREBP-2 showed consistent changes in transcription and translation. These results suggest that SCAP may not be a target of curcumin. As S1P protein levels decreased after curcumin treatment, curcumin may inhibit the expression of SREBP-2 by inhibiting the expression of S1P.
Several other studies have demonstrated inconsistency between SREBP-2 transcription and translation levels. Field et al [17] showed in rat liver cells that increased cholesterol in Caco-2 cells inhibited proteolysis of SREBP-2. The mSREBP-2 was decreased but pSREBP-2 levels were not significantly changed, and this process was accompanied by inhibition of SREBP-2 gene expression. Liu et al [10] reported that the change of SREBP-2 protein was not statistically significant after curcumin treatment compared with controls. Sato et al [18] found that the SREBP-2 gene includes a Sterol Regulatory Element (SRE) identical to the one on the promoter sequence of the human LDL receptor, thus SREBP-2 may regulate its own expression through modulating sterol levels. When the proteolytic process of SREBP-2 was inhibited, mSREBP-2 transcription factor levels decreased, which affected the expression of cholesterol metabolism related proteins and also the expression of SREBP-2. These results may explain why the expression of SREBP-2 mRNA does not follow the protein level.
Some studies have reported downregulated SREBP-2 upon curcumin treatment[9, 11, 12]. However, these studies do not show the molecular weight of the protein markers in their figures, making it hard to determine the molecular weight of the SREBP-2 detected in their western blots. Thus, the detected protein may be mSREBP-2 rather than pSREBP-2. Liu et al [10] studied the function of curcumin using antibodies against SREBP-2 purchased from Santa Cruz which have only been used to detect pSREBP-2. Our results indicate that curcumin first affects the SREBP-2 proteolysis process rather than inhibiting its protein expression. Protein expression inhibition may be a long-term effect. The inhibitory effect of curcumin on the transcription of SREBP-2 mRNA persisted long-term, with decreased mRNA of the pSREBP-2 detected after 96 hours treatment (Fig. 2b). When SREBP-2 proteolysis is inhibited, pSREBP-2 may be degraded by other pathways, and its gene expression may remain continuously inhibited, eventually leading to down-regulation of the pSREBP-2 protein expression. This mechanism still requires further experimentation to fully understand.
The exact mechanism for how curcumin reduces the hydrolysis of SREBP-2 and deceases mSREBP-2 protein levels through inhibiting S1P expression remains unknown. However, we conclude here that the mechanism of curcumin treatment on SREBP-2 is not through short-term inhibition of protein expression, although curcumin can indeed inhibit the transcription of SREBP-2 mRNA long-term. Thus, it appears that curcumin plays an important role via inhibiting the activity of SREBP-2 rather than directly affecting its expression of gene and protein.