Surfactant Protein A Activation of Atypical Protein Kinase C ζ in IκB-α-Dependent Anti-Inflammatory Immune Regulation

Abstract

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-κB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IκB-α, the primary regulator of NF-κB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IκB-α knockout/IκB-β knockin mice, SP-A fails to inhibit LPS-induced TNF-α production and p65 nuclear translocation, confirming a critical role for IκB-α in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) ζ as a pivotal upstream regulator of SP-A-mediated IκB-α/NF-κB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCζ−/− mice. SP-A transiently triggers aPKCThr410/403 phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IκB-α by SP-A critically depends on PKCζ activity, and thus attribute a novel, stimulus-specific signaling function to PKCζ in SP-A-modulated pulmonary immune response.

Pulmonary Surfactant Protein A Enhances Endolysosomal Trafficking in Alveolar Macrophages through Regulation of Rab7

“…Pharmacological inhibition and transient silencing of PKCz in RAW264.7 cells abolished the effects of SP-A on Rab7 stabilization. In line with this finding, it has been shown previously that PKCz inhibition/knockout had a significant negative effect on SP-A-mediated signaling events (38), pointing to an essential role for PKCz in SP-A-specific modulation of AM function. In contrast, SP-A-enhanced Rab7b stabilization was wortmannin insensitive, and coimmunoprecipitation experiments failed to show an SP-Aenhanced interaction of Rab7b with PKCz, suggesting a signaling pathway distinct from PI3K/PKCz for SP-A-enhanced Rab7b availability.…”

“…This is in contrast with a previous study (77) Several in vitro and in vivo studies have demonstrated that phosphatidylinositol-3,4,5-triphosphate directly activates the atypical PKCs (z, l/i) in a PI3K-dependent manner (78)(79)(80). It was shown that SP-A induces a rapid activation of PKCz in primary rat AM (38) and a rapid membrane localization of PKCz in human myometrial cells (53). A role in regulating vesicle trafficking has been implicated for the atypical PKCs by modulating cytoskeleton reorganization via Rho and Rac small GTPases (56,57) or by direct interaction with Rab2 (81).…”

“…After stimulation, cells were washed with 500 ml cold PBS, scraped off, and lysed in Laemmli buffer for 30 min at 4˚C to prepare whole-cell lysates (WCLs). For the preparation of cytosolic extracts, subcellular fractionation was performed as described previously (38). For experiments investigating proteins with low expression levels, WCLs were prepared.…”

Role of clathrin-mediated endocytosis of surfactant protein A by alveolar macrophages in intracellular signaling

“…After the cells were exposed to the experimental conditions, nuclear extracts were prepared and analyzed as described previously (50). The activity of NF-B in the nuclear extracts was determined by EMSA.…”

“…SP-A-enhanced aPKC kinase activity in AM has been shown previously to be critically involved in SP-A-mediated IB-␣ stabilization (50). We next investigated SP-A-induced aPKC kinase activity in the presence of Aman, PAO, CPZ, and CytoD in an in vitro immunocomplex kinase assay as described in MATERIALS AND METHODS (Fig.…”