The development of bleomycin-induced pulmonary fibrosis in rats was studied over a period of 21 d after an intratracheal instillation of bleomycin. The expression of three small proteoglycans (biglycan, decorin, and fibromodulin), collagen III and TGF-,B1 was studied by RNA-transfer blot analysis. The proteoglycans were also studied by SDS-polyacrylamide gel electrophoresis and Western blots.TGF-fi1 mRNA increased threefold already on day 3 and remained elevated until day 10. After the increase of TGF-,81 mRNA the messages for biglycan and collagen III steadily increased to reach a maximum 10 d after bleomycin instillation. The mRNA for biglycan increased maximally fourfold and that of collagen III 2.5-fold. Decorin mRNA, in contrast to biglycan decreased and reached 20% of control on day 10. The message for fibromodulin remained constant throughout the study period.The amounts of biglycan and decorin in the tissue changed in accordance with the mRNA levels. The results corroborate and extend previous in vitro studies concerning the effect of TGF-,i1 on the metabolism of small proteoglycans and show that these macromolecules are regulated differently also in vivo. The marked alterations of biglycan and decorin during the development of fibrosis suggests that these proteoglycans have a regulating role in this process. (J.

Transforming growth factor-␤ 1 (TGF-␤ 1 ) induces ␣-smooth muscle actin (␣-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased ␣-SMA expression. Because TGF-␤ 1 is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-␤ 1 alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [ 35 S]methionine, followed by protein separation on twodimensional gel electrophoresis, displayed ϳ2500 proteins in the pI interval of 3-10. Treatment of TGF-␤ 1 led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-␤ 1 , and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-␤ 1 induces not only ␣-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of ␣-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.

Inflammation is a response that has evolved over millions of years to become an extremely complex process. This complexity reflects the host's need to deal effectively with a wide variety of potentially injurious agents, as well as the need to incorporate an adequate set of checks and balances. An inappropriately checked response, which occurs rarely, results in disease, either acute or chronic. However, in most instances, inflammation is a beneficial response, essential for survival. Inflammation comprises an extensive network of cellular interactions implemented by an overwhelming number of molecules. One category of signal includes soluble products, such as neuropeptide, lipid mediators, cytokines and growth factors, most of which can be produced by inflammatory/haemopoietic cells. However, resident structural cells can also produce many of these products and, on this basis only, fibroblasts, epithelial, endothelial and smooth muscle cells should be considered as active contributors to the regulation of the inflammatory response. Extracellular matrix (ECM) proteins comprise another category of signals. Whilst the most recognized activities of these proteins are those concerned with providing structural tissue integrity, it is clear that they also have powerful inductive effects. Indeed, ECM proteins can influence the shape, movement and state of activation of inflammatory cells in the tissue. Recent evidence indicates that these signals may also play substantial roles in homing of inflammatory cells to certain sites and in the handling of a number of cytokines and growth factors. In so far as fibroblasts are the main producers of ECM proteins, these new data establish an indirect but important role for fibroblasts in the regulation of the inflammatory response.

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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.

Human skin fibroblasts were exposed to 3 anti-inflammatory steroids in order to study their effects on the glycosaminoglycan metabolism. The potent glucocorticoids, fluocinolone acetonide and budesonide, even at low concentrations strongly reduced the accumulation of hyaluronic acid and sulfated glycosaminoglycans in the medium, at the cell surface, and in the cells. Hydrocortisone had considerably less effect. The 3 compartments were not influenced to the same extent and the least inhibition was noted in the cell surface pool. Dermatan sulfate was decreased to the same relative extent in all 3 compartments, while hyaluronic acid and heparan sulfate were specifically retained at the cell surface, explaining why this compartment was less affected than the others. Dermatan sulfate was studied in more detail regarding effects on its copolymeric structure. Glucocorticoid treatment changed the uronosyl composition of the polysaccharides so that a relative decrease of glucuronic acid residues and a relative increase of iduronic acid residues were noted. This change was most evident in dermatan sulfate of the medium and of the cell surface. Thus, glucocorticoid treatment not only reduces the quantity of various glycosaminoglycans but also changes the distribution, the relative proportion, and the structure of connective tissue proteoglycans. These effects probably contribute to the development of skin atrophy, which often is observed after long-term treatment with potent glucocorticoids.

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