Ca2+/Calmodulin-Dependent Kinase Kinase α Is Expressed by Monocytic Cells and Regulates the Activation Profile

Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca2+/calmodulin-dependent kinase kinase α (CaMKKα) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKα by mass spectrometry and Western analysis. The function of CaMKKα in monocyte activation was examined using the CaMKKα inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKα, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKα to the nucleus. Finally, to further examine monocyte activation profiles, TNFα and IL-10 secretion were studied. CaMKKα inhibition attenuated PMA-dependent IL-10 production and enhanced TNFα production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKα in the differentiation of monocytic cells

Diversity of Microbial Sialic Acid Metabolism

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals

The CaMKKα inhibitor STO-609 enhances PMA-dependent up-regulation of CD86 but blocks CD11b expression and CaMKKα nuclear localization.

<p>(A and B) U937 cells were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in panel A and CaMKKα was quantified by Western analysis of nuclear lysates. Results are representative of three independent experiments. (D) U937 cells were treated as in panel A and ERK1/2 phosphorylation (ERK-p) and mass (ERK) were measured by Western analysis in whole cell lysates. Results are representative of three independent experiments.</p

CaMKKα inhibition enhances production of TNFα while reducing that of IL-10.

<p>(A and B) U937 cells (1×10⁶ cells/treatment) were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. TNFα and IL-10 concentrations were quantified from culture supernatants by cytokine assays. Results significantly different from PMA at α  = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM.</p

PMA induces CaMKKα nuclear localization.

<p>(A) U937 cells were treated with (+) or without (−) 100 nM PMA for 48 h and nuclear lysates were incubated with paramagnetic beads were as the solid phase. After extensive washing, proteins were resolved and visualized by SDS-PAGE and silver staining. Results are representative of three independent experiments. (B) U937 cells were treated and nuclear lysate affinity chromatography was performed as in panel A. CaMKKα mass was quantified by Western analysis. Results are representative of three independent experiments. (C) U937 cells were treated as in panel A. Affinity chromatography of whole cell lysates was performed as indicated. CaMKKα mass was quantified by Western analysis. Results are representative of three independent experiments.</p

Inhibition of CaMKKα by siRNA enhances PMA-dependent up-regulation of CD86 but decreases CD11b expression.

<p>(A and B) Cells were treated with 0.25 µg of either CaMKKα siRNA or scramble siRNA control prior to stimulation with 100 nM PMA for 48 h. Surface expression of CD11b and CD86 was quantified by flow cytometry. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in A and CaMKKα protein expression was quantified by intracellular flow. Results significantly different from control at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM.</p

The PKC inhibitor Bisindolylmaleimide inhibits PMA-dependent upregulation of CD86/CD11b expression.

<p>(A & B) U937 cells were pretreated with or without 1 µM bisindolylmaleimide for 15 min prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM.</p