Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells

Abstract

Summary 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 HS1 m 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.Keywords: glucocorticoid; heparan sulphate; adenocarcinoma; oncostatin M; interleukin 6; paracrine; fibroblast Differentiation of the mature phenotype in alveolar type II pneumocytes is marked by the production of lipid pulmonary surfactant (PS) and surfactant-associated proteins (Hawgood and Clements, 1990) and by an increase in alkaline phosphatase (AP) activity (Edelson et al, 1988). Perinatal induction of differentiation is mediated by glucocorticoids and a paracrine factor, or factors, from mesenchymally derived cells in the lung (Caniggia et al, 1991). In vitro, the synthesis of pulmonary surfactant and alkaline phosphatase can be induced in A549, a cell line derived from a human lung adenocarcinoma (Giard et al 1972), of reputedly type II cell origin (Lieber et al, 1976), by conditioned medium from lung fibroblasts (CM) (Speirs et al, 1991). Whereas induction of PS synthesis by CM was enhanced by treating the fibroblasts with dexamethasone (DX) before and during the conditioning process, DX was not required for the action of the CM on the A549 cells (Speirs et al, 1991). It is, however, required for the induction of alkaline phosphatase by CM and by several cytokines, such as oncostatin M (OSM), interleukin 6 (IL-6), and interferons a and [3, and by insulin (McCormick et al, 1995;McCormick and Freshney, 1996). OSM has been fou...

Hyaluronic acid production and CD44 expression in cultured dermal fibroblasts of patients with non-insulin-dependent diabetes mellitus with and without chronic ulcers on the lower extremity

“…Confluent cultures of dermal fibroblasts were incubated in EMEM supplemented with 5% FCS and 5 µCi/ml 3 H‐glucosamine (D‐[1– 3 H]‐glucosamine [3.15 Ci/mmol]; Amersham Pharmacia Biotech, Uppsala, Sweden). The determination of 3 H‐glucosamine incorporation into pericellular or secreted HA was carried out as described by us earlier with small modifications 13 . Briefly, the conditioned medium was aspirated and pooled.…”

Activity of growth factors in the IL-6 group in the differentiation of human lung adenocarcinoma

“…This suggests that the major active component of CM is neither IL-6 nor OSM, although it is acknowledged that native IL-6 or OSM in conditioned medium may be in a different form from the recombinant cytokines to which the antibodies were raised, e.g. bound to a cofactor such as a proteoglycan (Yevdokimova and Freshney, 1997) or by post-translational modification. However, CM, in the presence of DX, induced both AP activity and SPA gene expression in H441 cells, while OSM and IL-6 induced SPA but not AP, although some AP-inducing activity was seen with IFN-α.…”

“…In the current system DX has been found to increase the synthesis of a fraction of heparan sulphate present in the medium of DX-treated A549 cells. This fraction is heparinase and heparitinase-sensitive and can replace DX in the induction of AP in A549 cells by CM, IL-6 and OSM (Yevdokimova and Freshney, 1997). CNTF, a member of the IL-6 family, has been shown to interact with matrix in the differentiation of type 2 astrocytes (Lillien et al, 1990), and heparan Induction of AP in lung fibroblast-conditioned medium (CM).…”

Glucocorticoid Effects on Proteoglycans and Glycosaminoglycans

“…Changes in syndecan-1 expression are associated with attenuated biosynthesis of Osteoprotegerin and osteoclast formation, identifying tumor-derived syndecan-1 as a novel positive regulator of osteoclastogenesis and a new player in the tumor–bone dialog [ 79 ]. DXM at a dose of 0.25 µM was also shown to induce biosynthesis of HS and HA in subconfluent A549 cells by 5–10-fold 72 h after DXM administration [ 80 ]. Moreover, DEX administration at a dose of 100 nmol/L significantly decreased expression of CSPG CD44 in GR-positive bladder cancer cells both in mRNA (by 69.7%, p < 0.05) and protein levels 48 h after the treatment, although GR silencing in these cells had the opposite effects [ 81 ].…”