title: Role of clathrin-mediated endocytosis of surfactant protein A by alveolar macrophages in intracellular signaling.
abstract: We recently provided evidence that anti-inflammatory macrophage activation, i.e., the inhibition of constitutive and signal-induced NF-kappaB activity by the pulmonary collectin surfactant protein (SP)-A, critically involves a promoted stabilization of IkappaB-alpha, the predominant inhibitor of NF-kappaB, via posttranscriptional mechanisms comprising the activation of atypical (a)PKCzeta. SP-A uptake and degradation by alveolar macrophages (AMphi) occur in a receptor-mediated, clathrin-dependent manner. However, a mutual link between endocytosis of and signaling by SP-A remains elusive. The aim of this study was to investigate whether clathrin-mediated endocytosis (CME) of SP-A by AMphi is a prerequisite for its modulation of the IkappaB-alpha/NF-kappaB pathway. The inhibition of clathrin-coated pit (CCP) formation and clathrin-coated vesicle (CCV) formation/budding abrogates SP-A-mediated IkappaB-alpha stabilization and SP-A-mediated inhibition of LPS-induced NF-kappaB activation in freshly isolated rat AMphi, as determined by Western analysis, fluorescence-activated cell sorting, confocal microscopy, and EMSA. Actin depolymerization and inhibition of CCP formation further abolished SP-A-mediated inhibition of LPS-induced TNF-alpha release, as determined by ELISA. In addition, SP-A-induced atypical PKCzeta activation was abolished by pretreatment of AMphi with CCV inhibitors as determined by in vitro immunocomplex kinase assay. Although CME is classically considered as a means to terminate signaling, our results demonstrate that SP-A uptake via CME by AMphi has to precede the initiation of SP-A signaling.
authors.affiliation: Department of Clinical Medicine, Division of Cellular Pneumology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Moulakakis
authors.forename: Cordula
authors.initials: C
authors.lastname: Stamme
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000502:physiology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D020672:I-kappa B Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000502:physiology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000502:physiology*; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D000199:Actins; D002966:Clathrin; D004247:DNA; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D018832:Molecular Chaperones; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D037662:Pulmonary Surfactant-Associated Protein A; D014409:Tumor Necrosis Factor-alpha; C000606206:Nfkbia protein, rat; C490392:Prkcz protein, rat; D000099221:Protein Kinase C zeta