Transforming growth factor‐β induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts

Abstract

Human embryonic skin fibroblasts were pretreated with transforming growth factor-p (TGF-4) for 6 h and then labeled with [35S]sulphate and ['H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains.The results showed that TGF-P increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400 -500 kDa) increased 5 -7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3 -4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-fl treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected.To investigate whether TGF-P also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-P-trcated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-fi treated cultures contained a larger proportion of Dglucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4-and 6-sulphation of the GAG chains. By the use of p-nitrophenyl fi-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-P-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-fitreated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control.These novel findings indicate that TGF-P affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAGsynthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, whch can affect cell growth and migration as well as organisation of matrix fibres. Extracellular matrices consist of a variety of macromolecules such as collagens, fibronectin, hyaluronan and various proteoglycans. The latter are complex macromolecules consisting of a core protein substituted with glycosaminoglycans Correspondence to G.

Modifications of proteoglycans secreted into the growth medium by young and senescent human skin fibroblasts

“…When treated with specific antisera, this band showed a positive reaction as decorin core protein with a typical doublet and a negative response as biglycan core protein (Fig. 3B, lanes c,f) [23][24][25].…”

Interleukin-1 upregulates decorin production by arterial smooth muscle cells.

“…Since PG synthesis is known to be regulated by several serum components, such as TGF-/3 1322 and PDGF, 21 confluency in medium containing 10% serum. This was replaced with media containing 0.1%, 2.0%, 5.0%, or 10.0% serum with or without 500 pg/mL IL-1 for 36 hours before isolation of RNA.…”

Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats.

“…Decorin has also been shown to bind TGF-,B ( 15). The production of these proteoglycans is differently regulated in vitro, where biglycan is stimulated by TGF-0 (16)(17)(18). The factor also induces synthesis of proteoglycans with altered copolymeric structure of their glycosaminoglycan side chains ( 18).…”

“…The identity ofthe cDNA was confirmed by partial sequence analysis (Westergren-Thorsson, G., unpublished observation). 32P-labeled probes were prepared using a random priming cDNA labeling kit (Amersham International, Amersham, United Kingdom) as described ( 17).…”

“…After electrophoresis, a part ofthe gel was stained with ethidium bromide to verify that equal amounts of RNA were applied in each well of the gel. The filters were hybridized with 32P-labeled cDNA probes overnight in 50% formamide at 42°C (17) and then sequentially washed with 2x SSC (0.3 M sodium chloride, 30 mM sodium citrate pH 7.0) containing 0.1% sodium dodecyl sulfate at 42°C and then with 0.2x SSC, 0.1% sodium dodecyl sulfate at 50°C.…”