A Critical Role for the Inducible Proteasomal Subunits LMP7 and MECL1 in Cytokine Production by Activated Murine Splenocytes

Abstract

Background and Purpose: The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits β1, β2, and/or β5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the ‘immunoproteasome’. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes. Methods: A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1–/– mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified. Results: The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rβ, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1. Conclusions: Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.

Proteasome protease mediated regulation of cytokine induction and inflammation

“…Collectively, these results underscore the importance of LMP subunits in the proteasome of macrophages and splenocytes [27]. We have established that different proteasome proteases regulate levels of cytokine gene expression and protein expression differentially.…”

“…Since LPS does not directly activate CD4+ T cells, we used phorbol-12-myristate-13-acetate (PMA)/ionomycin as an agonist, thus bypassing the receptor. LMP7/LMP10-null splenocytes exhibited reduced levels of gene expression of IL-10, IL-4, IFN-γ, IL-2Rβ t-bet and GATA-3 (transcription factors) in response to agonists, whereas, induction of IL-13, IL-2, TNF-α and IL-2Rα was essentially normal in these knockout mice as compared to wild-type mice, and were found to be LMP-independent [27] and may therefore be X, Y, Z- dependent. Lactacystin (which predominantly inhibits the chymotrypsin-like activity of the proteasome) pretreatment of LMP7/LMP10-null splenocytes, followed by PMA/ionomycin, reduces gene expression of IL-2, IFN-γ IL-4, IL-13, TNF-α(cytokines), IL-2Rα and IL-2Rβ (receptors) t-bet and GATA-3 (transcription factors).…”

“…Lactacystin (which predominantly inhibits the chymotrypsin-like activity of the proteasome) pretreatment of LMP7/LMP10-null splenocytes, followed by PMA/ionomycin, reduces gene expression of IL-2, IFN-γ IL-4, IL-13, TNF-α(cytokines), IL-2Rα and IL-2Rβ (receptors) t-bet and GATA-3 (transcription factors). In contrast, levels of expression of IL-10 (anti-inflammatory cytokine) were increased upon pretreatment of cells with lactacystin in splenocytes from null as compared to controls [27]. Thus, as with macrophages, production of subsets of inflammatory cytokines by stimulated T cells, is influenced by proteasome subunit composition, or differential protease activities of cellular proteasomes.…”

The Immunoproteasome in oxidative stress, aging, and disease

“…Studies have shown that the Immunoproteasome plays a role in cytokine production in response to inflammation or infection, and cells or animals deficient in Immunoproteasome subunits have decreased cytokine signaling (Muchamuel et al, 2009, Arima et al, 2011, Kitamura et al, 2011, Liu et al, 2012, Rockwell et al, 2012). Cytokines that have been shown to be regulated by the Immunoproteasome include interferon- γ, IL-2, IL-4, IL-10, and IL-23, and cells from LMP7 −/− MECL1 −/− knockout mice have been used to show that lack of the Immunoproteasome also causes decreased expression of two cytokine transcription factors, GATA3 and t-bet (Rockwell et al, 2012, Muchamuel et al, 2009).…”

“…Cytokines that have been shown to be regulated by the Immunoproteasome include interferon- γ, IL-2, IL-4, IL-10, and IL-23, and cells from LMP7 −/− MECL1 −/− knockout mice have been used to show that lack of the Immunoproteasome also causes decreased expression of two cytokine transcription factors, GATA3 and t-bet (Rockwell et al, 2012, Muchamuel et al, 2009). Taken together, these findings suggest that the Immunoproteasome not only plays a role in responding to inflammation, but also participates in activating the inflammatory cytokines, as well.…”