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Biosynthesis of the third component of complement (C3) by the human monocytic-cell line U-937. Induction by phorbol myristate acetate.

ABSTRACT: Phorbol myristate acetate (PMA)-stimulated human monocyte-like cells (U-937) were found to synthesize the third component of complement (C3), as shown by enzyme-linked immunosorbent assay and immunoprecipitation from [35S]methionine-labelled culture supernatants. C3 synthesis occurred at a rate of about 160 ng of C3/24 h per 10(6) cells on day 7 after addition of PMA; it was blocked by cycloheximide treatment and was restored after removal of the inhibitor. SDS/polyacrylamide-gel-electrophoretic analysis of the immunoprecipitated protein showed that the size and subunit structure of the newly synthesized C3 were identical with those of plasma C3, and that a single-chain intracellular precursor was present in the cell lysates. Haemolytic assays showed that the synthesized C3 fully expressed functional activity in early culture within 4 h. After longer culture, a loss of haemolytic activity was observed. The possibility that newly secreted C3 is cleaved by U-937 cells themselves was suggested.

SUBMITTER: Bengio S

PROVIDER: S-EPMC1147344 | biostudies-other | 1986 Nov

REPOSITORIES: biostudies-other

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Phorbol 12-Myristate 13-Acetate-Induced Changes in Chicken Enterocytes.

Project description:Increased intestinal epithelial permeability has been linked to many enteric diseases because it allows easy access of microbial pathogens and toxins into the system. In poultry production, the restrictions in the use of antibiotic growth promoters have increased the chances of birds being susceptible to different enteric diseases. Thus, understanding the mechanisms which compromise intestinal function is pertinent. Based on our previous observation which showed the primary chicken enterocytes in culture undergoing dystrophic changes on treatment with phorbol myristate acetate (PMA), we surmised that this model, which appeared to mimic increased intestinal permeability, may help to understand the mechanisms of this problem. As genomic and proteomic changes are associated with many physiological and pathological problems, we were interested to find whether certain proteomic changes underlie the morphological alterations in the enterocytes induced by PMA. We exposed primary enterocyte cultures to a sub-lethal concentration of PMA, extracted the proteins, and analyzed by mass spectrometry for differentially regulated proteins. Our results showed that PMA affected several biological processes which negatively affected their energy metabolism, nuclear activities, and differentially regulated the levels of several stress proteins, chaperon, cytoskeletal, and signal transduction proteins that appear to be relevant in the cause of enterocyte dystrophy. Phorbol myristate acetate-affected signal transduction activities also raise the possibilities of their increased susceptibility to pathogens. The changes in enterocyte integrity can make intestine vulnerable to invasion by microbial pathogens and disrupt gut homeostasis.

| S-EPMC6463336 | biostudies-literature

Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937.

Project description:Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.

| S-EPMC1138841 | biostudies-other

Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

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| S-EPMC153529 | biostudies-literature

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| S-EPMC4134517 | biostudies-literature

The separation of functionally distinct forms of the third component of human complement (C3).

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2015-11-09 | GSE67264 | GEO

Gene expression analysis of U937 cells during the differentiation by phorbol 12-myristate 13-acetate (PMA)

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Expression profiling of exposure to phorbol myristate acetate - PMA and FOXK2 depletion with siRNA

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2011-11-03 | E-MEXP-3107 | biostudies-arrayexpress

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| S-EPMC4332032 | biostudies-literature

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