Dataset Information

Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

ABSTRACT: Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.

SUBMITTER: Nagase H

PROVIDER: S-EPMC1152245 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundMacrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.MethodsImmunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM.ResultsSM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM.ConclusionsThese findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.

Dataset Information

Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets