Abstract
Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.
Highlights
We have shown that oncostatin M (OSM) increases the expression of Received 14 August 1996 Revised 20 November 1996
We have shown that there is a marked stimulation of [3H]glucosamine ([3H]GLN) incorporation, in the medium of subconfluent A549 cells, and that a fraction eluted from DEAE in 0.35 M sodium chloride, corresponding to an early-eluting fraction of heparan sulphate (HS), substitutes for DX in the induction of alkaline phosphatase (AP) by conditioned medium (CM) and cytokines, such as interleukin 6 (IL-6) and OSM
P3 corresponded to the position of the unlabelled heparan sulphate, the bulk of which eluted at 0.4 M sodium chloride
Summary
Technologies) and supplemented with 10% fetal bovine serum (Globepharm) (FIO:DMEMIOFB) They were propagated in conventional plastic flasks (Bibby Sterilin or Nunc) and subcultured weekly, seeding at 104 cells ml-' (2 x 103 cells cm-2), and with one intermediate medium change. LF1 13 cells were grown to confluence, the serum reduced to 1%, and the confluent monolayer was maintained for 8 days, at which time the serum was removed and serum-free F1O:DMEM (SF) added for a further 3 days (McCormick et al, 1995). It was frozen and thawed at least once and filtered through a 0.22-gm filter (Millex GV, Millipore) before use
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