Murine macrophages are activated by interferon-γ (IFNγ) and/or TLR agonists such as bacterial endotoxin (LPS) to express an inflammatory (M1) phenotype characterized by expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as TNF-α and IL-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα) dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A2A receptor (A2AR) agonists act synergistically with TLR2, 4, 7 and 9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d”. Adenosine signaling suppresses TLR-dependent expression of TNF-α, IL-12, IFN-γ and several other inflammatory cytokines by macrophages, and induces expression of VEGF and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα−/− mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10 and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α) or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. Use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Non-activated macrophages express low levels of A(2A)Rs and lipopolysaccharides (LPS) upregulates A(2A)R expression in an NF-κB-dependent manner. The murine A(2A)R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5' untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A(2A)Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402-417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309-320), STAT1 (526-531) and AP2 (566-569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582-588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A(2A)R expression in macrophages in response to inflammatory stimuli.

Background Protein kinase C (PKC) serves as the receptor for tumor-promoting phorbol esters, which are potent activators of conventional (c) and novel (n) PKCs. We recently showed that these activators induced selective upregulation of PKCη in breast cancer cells. The objective of this study is to understand unique regulation of PKCη and its importance in breast cancer. Methods The levels of PKC isozymes were monitored in breast cancer cells following treatment with inhibitors of kinases, proteasome and proteases by Western blotting. PKCε was introduced by adenoviral delivery. PKCη and PDK1 were depleted by siRNA silencing. Cell growth was determined by the MTT or clonal assay. Results The general PKC inhibitors Gö 6983 and bisindolylmaleimide but not cPKC inhibitor Gö 6976 led to substantial PKCη downregulation, which was partly rescued by the introduction of nPKCε. Inhibition of phosphoinositide-dependent kinase-1 (PDK1) by Ly294002 or knockdown of PDK1 also led to downregulation of basal PKCη but had no effect on PKC activator-induced upregulation of PKCη. Proteasome inhibitors blocked PKCη downregulation triggered by PDK1 inhibition/depletion but not by Gö 6983. PKCη level increased in malignant but not in non-tumorigenic or pre-malignant cells in the progressive MCF-10A series associated with activated PDK1, and knockdown of PKCη inhibited breast cancer cell growth and clonogenic survival. Conclusion Upregulation of PKCη contributes to breast cancer cell growth and targeting either PKCε or PDK1 triggers PKCη downregulation but involves two distinct mechanisms. General significance The status of PKCη may serve as a potential biomarker for breast cancer malignancy.

Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, we have investigated the molecular mechanism of PKCη regulation. Several PKC activators, including phorbol 12, 13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate and indolactam V caused upregulation of PKCη whereas the general PKC inhibitor Gö 6983, but not the conventional PKC inhibitor Gö 6976 led to the downregulation of PKCη. Upregulation of PKCη was associated with an increase in phosphorylation of PKCη. Silencing of phosphoinositide-dependent kinase-1, which phosphorylates PKCη at the activation loop, failed to prevent PKC activator-induced upregulation of PKCη. Knockdown of PKCε but not PKCα inhibited PKC activator-induced upregulation of PKCη. Thus, our results suggest that the regulation of PKCη is unique and PKCε is required for the PKC activator-induced upregulation of PKCη.

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