The primary objectives in the present study were to further evaluate several naturally-occurring proteasome inhibitors for their capacity to suppress inflammatory processes, and to define mechanisms responsible for these anti-inflammatory effects. Anti-inflammatory properties of the proteasome inhibitors were evaluated in macrophages derived from several sources (e.g. the RAW 264.7 cell line, and thioglycolate-elicited peritoneal macrophages from four strains of mice (C57BL/6, BALB/c, double knockout LMP7/MECL-1-/- and PPAR-α-/-). As a result of these studies we have identified several naturally-occurring proteasome inhibitors that could potentially decrease levels of inflammatory cytokines and NO that may contribute to the development of diseases associated with ageing.
First, we demonstrated that dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin are potent inhibitors of chymotrypsin-like activity of 20S rabbit muscle proteasomes, and that (-) Corey lactone and amiloride enhanced this activity. We also demonstrated that dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin inhibited the secretion of TNF-α and NO production by LPS-stimulated RAW 264.7 macrophage like murine cell cultures. Further, levels of NF-κB within the nucleus were decreased, whereas cellular P-IκB levels were increased, by pre-treatment of LPS-stimulated RAW 264.7 cells with dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin.
NF-κB is maintained in an inactive state in the cytoplasm of cells when it is bound to IκB. LPS induces a series of events that results in phosphorylation and ubiquitination of IκB with subsequent degradation by the proteasome. These actions result in NF-κB activation, translocation to the nucleus, and increased transcription of several genes encoding pro-inflammatory cytokines. Consequently, the capacity of dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin to inhibit proteasome activity in conjunction with their capacity to increase cellular levels of P-IκB and decrease nuclear translocation of NF-κB, suggests that the mechanism by which these agents suppress production of TNF-α, and NO involves decreased degradation of ubiquinated P-IκB by the proteasome, resulting in depressed translocation of NF-κB to the nucleus. Therefore, ultimately, these proteasome inhibitors suppress production of TNF-α, and NO, and exert their anti-inflammatory effects by inhibiting NF-κB, activation.
This conclusion was further supported by experimental testing of these inhibitors (dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin) in LPS-stimulated thioglycolate-elicited peritoneal macrophages derived from four different strains of mice. All inhibitors significantly inhibited LPS-induced secretion of TNF-α by macrophages derived from C57BL/6 and BALB/c mice (Figure 9A). Although, macrophages derived from C57BL/6 mice compared to BALB/c mice yielded slightly better (20%) inhibition in LPS-stimulated secretion of TNF-α (Figure 9A). In marked contrast to the results attained with C57BL/6 and BALB/c mice, TNF-α, secretion was essentially unaffected by treatment of LPS-stimulated macrophages derived from LMP7/MECL-1-/- knockout mice with δ-tocotrienol, riboflavin, and quercetin (9B). Similarly, these compounds failed to suppress TNF-α secretion by LPS-stimulated macrophages derived from PPAR-α-/- knockout mice (since activation of PPAR-α-/- mice normally reduced inflammation); δ-tocotrienol and riboflavin treatment actually enhanced TNF-α secretion (Figure 9B). In marked contrast to the observations with TNF-α, δ-tocotrienol, riboflavin, and quercetin suppressed NO production by LPS-stimulated macrophages from C57BL/6, BALB/c, LMP7/MECL-1-/-, and PPAR-α-/- knockout mice (Figure 10A, B). The effects of these proteasome inhibitors on mRNA production by LPS-stimulated macrophages were generally consistent with at the protein levels of TNF-α, secretion and NO production.
Our previously published studies strongly support the concept that proteasomes are key regulators of LPS-stimulated inflammatory signaling pathways (3,4,6-8). Proteolytic activity of proteasomes is mediated by the 20S proteasomes, a hollow, cylindrical multi-protein complex consisting of three proteolytic subunits, X, Y, and Z, with chymotrypsin-like, trypsin-like, and post-glutamase activities, respectively. A variety of inflammatory stimuli induce alterations in newly assembled "immuno-proteasomes" in which X, Y and Z subunits are partially replaced by LMP7, LMP2, and MECL-1, respectively.
We previously demonstrated that low doses of lactacystin, which suppresses primarily chymotrypsin-like activity of the proteasome, potently suppressed production of NO, but not TNF-α by LPS stimulated macrophages. We further demonstrated that in order to suppress TNF-α secretion by LPS-stimulated macrophages with proteasome inhibitors, both chymotrypsin-like and trypsin-like proteasome activities must be suppressed (22). Subsequent experiments with proteasome subunit knockout (LMP7-/-, LMP2-/-, MECL-1-/-, and LMP7/MECL-1-/-) revealed that NO production by LPS-stimulated peritoneal macrophages was markedly reduced in LMP7-/-, LMP2-/-, MECL-1-/-, and LMP7/MECL-1-/-knockout mice; TNF-α production, in contrast, was not markedly affected by any of these knockout genotypes .
In the current study, the capacity of the investigated proteasome inhibitors to inhibit TNF-α secretion by LPS-stimulated macrophages from several sources (i.e. the RAW 264.7 cell line, and peritoneal macrophages from C57BL/6 and BALB/c mice) supports the conclusion that these inhibitors suppress both chymotrypsin-like and trypsin-like activities of the proteasomes, since both of these activities must be suppressed in order to inhibit TNF-α, secretion. These conclusions were supported by our analysis of the effects of these inhibitors on proteolytic activity of the proteasome.
The capacity of δ-tocotrienol, riboflavin, and quercetin to block TNF-α secretion in C57BL/6 and BALB/c, but not in LMP7/MECL-1-/- mice, is an intriguing result. LPS-stimulation of macrophages from C57BL/6 and BALB/c mice would be expected to induce production of immunoproteasomes in which X, Y and Z subunits were partially replaced by LMP2, LMP7 and MECL-1-/-. The × and Z components of LPS-stimulated macrophages from LMP7/MECL-1-/- mice, however, cannot be replaced by LMP7 and MECL-1-/-. The macrophages from knockout mice when induced produce robust amounts of TNF-α. Thus, the differential capacity of δ-tocotrienol, riboflavin, and quercetin to inhibit TNF-α secretion by LPS-stimulated macrophages from C57BL/6 and BALB/c vs LMP7/MECL-1-/- knockout mice would appear to be attributable to a differential susceptibility of × and Z vs LMP7 and MECL-1 proteasomal subunits to inhibition by these inhibitors.
Presumably δ-tocotrienol, riboflavin, and quercetin are potent inhibitors of LMP2, LMP7 and MECL-1-/- subunits of mouse immunoproteasomes, with comparatively lesser suppressive effects on X, Y, and Z subunits of constitutively expressed proteasomes. Thus, the chymotrypsin-like and trypsin-like activities of normal immunoproteasomes (containing LMP2, LMP7 and MECL-1-/-) from C57BL/6 and BALB/c mice would be suppressed by δ-tocotrienol, and quercetin, resulting in decreased TNF-α secretion. In contrast, the chymotrypsin-like and trypsin-like activity of proteasomes (containing X, Y, Z, and LMP2) from LMP7/MECL-1-/- knockout mice would not be suppressed by δ-tocotrienol, riboflavin, and quercetin, resulting in normal TNF-α secretion.
The finding that δ-tocotrienol, riboflavin, and quercetin inhibited NO production by LPS-stimulated macrophages from C57BL/6, BALB/c, LMP7/MECL-1-/-, and PPAR-α-/- mice was also quite interesting; the LMP7/MECL-1-/- genotype mice did not affect susceptibility to inhibition by these compounds. These macrophages from knockout mice do not induce very much NO, therefore IFN-γ had to be added along with LPS to induce NO in cells that have X, Y, Z, and LMP2 subunits .
PPAR-α-/- knockout mice have exaggerated inflammatory responses to a variety of stimuli, because activation of PPAR-α leads to anti-inflammatory effects . The mechanisms leading to these exaggerated inflammatory responses are not clearly understood, but are believed to be at least partially attributable to increased NFκB activity [24, 25]. Consequently, one would expect TNF-α secretion by LPS-stimulated macrophages from PPAR-α-/- knockout mice to be highly up-regulated with respect to TNF-α secretion and relatively resistant to inhibition by proteasome inhibitors that degrade IκB, and decrease NF-κB activity. Therefore, we found that δ-tocotrienol, riboflavin, and quercetin failed to suppress TNF-α, secretion by LPS-stimulated macrophages from PPAR-α-/- knockout mice. In fact TNF-α secretion was substantially enhanced by riboflavin and δ-tocotrienol. A clearer explanation of these results will be dependent on further elucidation of interactions between PPAR-α, and ubiquitin proteasome pathways .
Our present results demonstrate that δ-tocotrienol, riboflavin, and quercetin are naturally-occurring potent proteasome inhibitors for the inhibition of NO production tested in vitro. These results also confirms earlier report that γ-tocotrienol blocked LPS-stimulated activation of NF-κB, and also blocked TNF-α induced phosphorylation and degradation of IκBα, through the inhibition of IκBα kinase activation . Tocotrienols have been shown to modestly inhibit or activate the proteasomal activity depending on its concentrations (Tables 1, ). Therefore, blocking the proteasomal activity with low doses of tocotrienols could potentially reduce inflammatory responses, but at high doses of tocotrienols may cause apoptotic cell death in cancer [9, 28]. Quercetin, on the other hand is a natural proteasome inhibitor that can affect several proteasomal activities.