Medium Of Fibroblasts Research Articles

Mechanism of Abnormal Elastin Gene Expression in the Pinguecular Part of Pterygia

Elastodysplasia and elastodystrophy are two known manifestations in the conjunctival, ie, pinguecular, part of pterygia. But the mechanisms are still not understood. The purpose of this study is to investigate the mechanism of enhanced elastin gene expression in fibroblasts from the pinguecular part of pterygia, which is related to abnormal elastic fiber expression in the pinguecular part of pterygia. Elastin in surgical specimens of normal conjunctiva and the pinguecular part of pterygia from age-matched patients was detected by immunohistochemical staining. Northern hybridization and quantification of radiolabeled tropoelastin were performed in conjunctival fibroblasts cultured under different doses of ultraviolet (UV) B irradiation, and in cultured pinguecular fibroblasts from pterygia. In vitro translation was also performed to analyze the tropoelastin production in rabbit reticulocyte lysate. The level of tropoelastin in reticulolysates from UV-treated conjunctival and pinguecular fibroblasts of pterygia was higher than in normal conjunctival fibroblasts. The coding sequence and 3'- untranslated region of tropoelastin mRNAs were amplified by reverse transcription-polymerase chain reaction, and mutations were checked by DNA sequencing. Immunohistochemical staining revealed elastin in pinguecular subepithelial connective tissues of pterygia, but not in normal conjunctiva. Tropoelastin mRNA levels were not elevated in cultured pinguecular or conjunctival fibroblasts with or without ultraviolet B irradiation. However, tropoelastin synthesis was enhanced in culture medium of pinguecular and UV-irradiated conjunctival fibroblasts, but not in normal conjunctival fibroblasts. Direct DNA sequencing revealed mutations in the 3'-untranslated region but not in the coding sequence of tropoelastin mRNA, in both pinguecular and UV-irradiated conjunctival fibroblasts. The increased expression of tropoelastin in pinguecular and UV-irradiated fibroblasts is not a result of increased levels of steady-state mRNA, but is a result of posttranscriptional modification of tropoelastin.

Vascular and cellular proteolytic activity in mice with α2-antiplasmin gene inactivation

Objective: To study the role of α2-antiplasmin (α2-AP), the main physiological plasmin inhibitor, in controlling vascular and cellular proteolytic activity.Materials: Arteries, organs and cell cultures derived from α2-AP-deficient (α2-AP–/–) mice or from their wild-type littermates (α2-AP+/+).Results: In serum-free conditioned medium of α2-AP+/+or α2-AP–/–skin fibroblasts, the time course (0–72 h) of PAI-1 antigen and of t-PA or u-PA antigen and activity production was similar. Activation of proMMP-9 (gelatinase B) upon addition of plasmin(ogen) to serum-free conditioned medium of fibroblasts was consistently detectable with α2-AP–/–but not with α2-AP+/+cells. In aorta and femoral arterial extracts of α2-AP+/+or α2-AP–/–mice, t-PA and u-PA activity levels were comparable, and fibrin zymography with cryosections did not reveal significant differences in fibrinolytic activity. In liver or kidney extracts of α2-AP+/+or α2-AP–/–mice, t-PA, u-PA, PAI-1 and plasminogen antigen levels were comparable; t-PA or u-PA activity was not detected in liver extracts and was present at comparable levels in kidney extracts.Activation of plasminogen to plasmin in solution by cell-associated plasminogen activator, and activation of cell-bound plasminogen by tcu-PA was comparable for fibroblasts of both genotypes.Conclusions: α2-AP does not play a crucial role in controlling vascular or cellular proteolytic activity in mice.

A factor implicated in the myogenic conversion of nonmuscle cells derived from the mouse dermis.

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein beta-galactoside binding protein (betaGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts. could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing betaGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.

Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity.

Bovine joint capsule was maintained in explant culture in the presence of bovine aggrecan monomer and it was shown that the aggrecan monomer was degraded. Amino-terminal sequence analysis of the resulting aggrecan core protein fragments revealed that the core protein was cleaved at five specific sites attributed to glutamyl endopeptidases referred to as aggrecanase activity. Fibroblast cultures were established from explant cultures of joint capsule and when these cells were exposed to aggrecan, cleavage of the core protein of aggrecan at the aggrecanase sites was observed. Inclusion of either retinoic acid or interleukin-1alpha in medium of either joint capsule explant cultures or fibroblast cultures did not increase the rate of cleavage of exogenous aggrecan present in the culture medium. When aggrecan monomer was incubated with conditioned medium from explant cultures of joint capsule maintained in medium, degradation could be detected after 10 min. After a 6-h incubation period the same fragments of aggrecan core protein were observed as those for tissue or cells incubated directly with aggrecan monomer. RT-PCR analysis of mRNA extracted from joint capsule fibroblasts showed that these cells express both aggrecanase-1 and -2 [ADAMTS-2 (Tang) and ADAMTS-5].

Proinflammatory cytokines differentially regulate hyaluronan synthase isoforms in fetal and adult fibroblasts

Background/Purpose: Fetal wound healing is a relatively scarless process that occurs in an hyaluronan-rich environment. Understanding the regulation of hyaluronan expression may provide insight into the process of fetal repair. Therefore, the purpose of this study was to compare the regulation of hyaluronan and hyaluronan synthase transcripts by the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in human adult and fetal fibroblasts. Methods: Hyaluronan deposited in the medium of untreated fibroblasts or fibroblasts treated with either IL-1β or TNF-α was determined by an assay utilizing iodine I 125-hyaluronan binding protein. HAS transcript levels were compared in using a ribonuclease protection assay. Results: IL-1β induced an increase in hyaluronan accumulation by both fetal and adult fibroblasts. In contrast, TNF-α induced higher levels of hyaluronan only in fetal fibroblasts. HAS-2 and HAS-3 transcript levels were constitutively expressed by both fetal and adult fibroblasts. Proinflammatory cytokines induced a differential increase in HAS-1 and HAS-3 transcript levels. Conclusions: Differential regulation was observed in hyaluronan accumulation and for HAS transcript levels in fetal and adult dermal fibroblasts. The muted response of fetal fibroblasts to cytokines may be relevant to the minimal inflammation associated with fetal repair. J Pediatr Surg 35:874-879. Copyright © 2000 by W.B. Saunders Company.

A novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer.

When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1-3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1-3 segment, and by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, alpha1-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1-3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo.

Growth factor-extracellular matrix synergy in the control of trophoblast invasion.

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin alpha5beta1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor beta1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.

Comparison of the Effects of Dimyristoyl and Soya Phosphatidylcholine Liposomes on Human Fibroblasts

The in vitro effects of prolonged exposure (8 days) of human skin fibroblasts to several concentrations of extruded dimyristoyl (dm-PC) and soya phosphatidylcholine (soya-PC) liposomes were compared. Prepared liposome suspensions were added to the fibroblast culture medium at phospholipid concentrations of 10, 50, 100, 200, and 300 mu M. Survival curves and values of 50% inhibitory concentration (IC50) and area under the curve (AUC) were used to compare the response of the fibroblasts to the two types of liposomes. The effect of the incorporation of vitamin E in the liposomal preparations also was determined. Fibroblasts showed greater sensitivity toward the soya-PC liposomes (IC50 = 150 mu M) than the dm-PC liposomes (IC50 = 212 mu M). The presence of vitamin E in the soya-PC liposomes led to a 1.9-fold increase in the IC50, while dm-PC liposomes containing vitamin E showed an IC50 that was 1.1 times higher than that shown by control vitamin-free liposomes. Soya-PC liposomes containing vitamin E at a molar ratio of 10:0.5 (phospholipid:vitamin)were best tolerated by the fibroblasts (IC50 > 300 mu M). It would appear that dm-PC liposomes are better tolerated by fibroblasts than those composed of soya-PC. However, the incorporation of vitamin E into the liposomes seems to reverse this effect, and it is the vitamin-containing soya-PC liposomes that are most compatible with the growth of fibroblasts in culture.

Initial Steps in Assembly of Microfibrils: FORMATION OF DISULFIDE-CROSS-LINKED MULTIMERS CONTAINING FIBRILLIN-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.

Alpha-thrombin stimulates expression of macrophage migration inhibitory factor in skin fibroblasts.

Macrophage migration inhibitory factor (MIF) is induced by various stimuli such as wounds and infection and regulates inflammatory and immunological responses. To date, we have found increased expression of MIF during the wound healing process in rat skin. Immunohistochemical analysis demonstrated enhanced expression of MIF in wound skin lesions. On the other hand, alpha-thrombin, a multifunctional serine protease, plays an important role in wound healing with regard to induction of inflammatory cytokines such as interleukin-6 (IL-6). Accordingly, we examined the effect of alpha-thrombin on MIF production in human skin fibroblasts. Alpha-thrombin significantly stimulated MIF secretion into culture medium of fibroblasts quantitated by an enzyme-linked immunosorbent assay (ELISA). Consistent with this, we observed the upregulation of MIF mRNA in response to alpha-thrombin by Northern blot analysis. Taken together, these results suggest that MIF produced by fibroblasts in response to alpha-thrombin plays an important regulatory role in wound repair.

Histopathological and Cellular Studies of a Case of Cutaneous Radiation Syndrome after Accidental Chronic Exposure to a Cesium Source

This study was designed for the histopathological, cellular and biochemical characterization of a skin lesion removed surgically from a young male several months after accidental exposure to cesium-137, with an emphasis on expression of transforming growth factor beta1 (TGFB1) and tumor necrosis factor alpha (TNFA) and the occurrence of apoptosis. Under a hypertrophic epidermis, a highly inhomogeneous inflammatory dermis was observed, together with fibroblastic proliferation in necrotic areas. Immunostaining revealed overexpression of TGFB1 and TNFA inside the keratinocytes of the hypertrophic epidermis as well as in the cytoplasm of the fibroblasts and connective tissue of the mixed fibrotic and necrotic dermis. Inside this dermis, the TUNEL assay revealed areas containing numerous apoptotic fibroblasts next to areas of normal viable cells. Overexpression of TGFB1 was found in the conditioned medium and cellular fractions of both hypertrophic keratinocytes and fibrotic fibroblasts. This overexpression lasted for at least three passages in tissue culture. The present observations were consistent with the central role of TGFB1 in the determination of chronic radiation-induced damage to the skin and a significant involvement of TNFA. In addition, programmed cell death appeared to take place during the remodeling of the mixed fibrotic and necrotic tissue.

A New Type of Carbohydrate-deficient Glycoprotein Syndrome Due to a Decreased Import of GDP-fucose into the Golgi

The fucosylation of glycoproteins was found to be deficient in a patient with a clinical phenotype resembling that of leukocyte adhesion deficiency type II (LAD II). While in LAD II hypofucosylation of glycoconjugates is secondary to an impaired synthesis of GDP-fucose due to a deficiency of the GDP-D-mannose-4, 6-dehydratase, synthesis of GDP-fucose was normal in our patient (Körner, C., Linnebank, M., Koch, H., Harms, E., von Figura, K., and Marquardt, T. (1999) J. Leukoc. Biol., in press). Import of GDP-fucose into Golgi-enriched vesicles was composed of a saturable, high affinity and a nonsaturable component. In our patient the saturable high affinity import of GDP-fucose was deficient, while import of UDP-galactose and the activity of GDPase, which generates the nucleoside phosphate required for antiport of GDP-fucose, were normal. Addition of L-fucose to the medium of fibroblasts restored the fucosylation of glycoproteins. We propose that this new form of carbohydrate-deficient glycoprotein syndrome is caused by impaired import of GDP-fucose into the Golgi.

Expression of Type XVI Collagen in Human Skin Fibroblasts: Enhanced Expression in Fibrotic Skin Diseases

Abundance of type XVI collagen mRNA in normal human dermal fibroblasts explanted from different horizontal layers was determined using RNase protection assays. Type XVI collagen mRNA level in the fibroblasts explanted from the upper dermis was greater than those of the middle and lower dermis. The antibody raised against the synthetic N-terminal noncollagenous region reacted with approximately 210 kDa collagenous polypeptide in the culture medium of fibroblasts. Immunohistochemical study of normal human skin demonstrated that the antibody reacted preferentially with the fibroblasts and the extracellular matrix in the upper dermis rather than those in the middle and lower dermis. Type XVI collagen mRNA level was elevated 2.3-fold in localized scleroderma and 3.6-fold in systemic scleroderma compared with keloid and normal controls. Immunofluorescent study revealed that an intense immunoreactivity with the antibody was observed in the upper to lower dermal matrix and fibroblasts in the skin of systemic scleroderma as compared with normal skin. The results suggest that expression of type XVI collagen, a member of fibril-associated collagens with interrupted triple helices, in human skin fibroblasts can be heterogeneous in the dermal layers and can be modulated by some fibrotic diseases.

Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin–angiotensin system

Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin-1 released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT 1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways.

Energy metabolism inhibition impairs amyloid precursor protein secretion from Alzheimer's fibroblasts

The present study investigates the influence of aglycemia and sodium azide (a Cytochrome c Oxidase inhibitor) on sAPP secretion from skin fibroblasts derived from sporadic AD patients and control subjects. Aglycemia reduced sAPP release in the medium of both AD and control fibroblasts to a similar extent after 2 h incubation. Treatment for 2 h with increasing azide concentrations (1 μM–100 mM) under glucose deprivation did not significantly affect sAPP secretion from control fibroblasts, but was able to significantly inhibit sAPP secretion from AD fibroblasts (maximal inhibition 51%). The failure of antioxidants like glutathione (GSH) or N-acetylcysteine (NAC) to antagonize the azide effect on AD fibroblasts and lipoperoxidation data seemed to rule out the possibility that oxidative stress could mediate the sodium azide effect on sAPP release from AD fibroblasts.

In vitro reconstitution of the tracheal epithelium.

We have developed a unique in vitro reconstitution system for tracheal epithelia of guinea pigs. In the system, a human amnion membrane was used as a basement membrane and the tracheal epithelial cells were cultured on the epithelial side of the membrane. Three weeks later, the tracheal fibroblasts were co-cultured on the serosal side of the amnion membrane and the culturing was continued for an additional 10 d. The morphology of the cultured epithelial cells consisted of a pseudostratified columnar ciliated epithelium from cuboidal ciliated epithelium during the last 10 d of the culture period. Epithelial cells included both goblet-like and basal cells. In addition, the frequency of each type of differentiated cells was almost identical to that of in vivo tracheas. Interestingly, the same results were obtained when the conditioned medium of the tracheal fibroblasts was used instead of the fibroblasts themselves. These results suggest that epithelial-mesenchymal interaction is likely involved in growth and differentiation of epithelial cells in vivo in a soluble factor(s)-mediated manner. As well as the epithelial cells, the fibroblasts also formed a multilayer during the last 10 d of co-culturing. This indicates that in vitro reconstitution of tracheal epithelia is achieved without addition of any exogenous growth or differentiation factors. The reconstitution system is shown to be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. Possible applications of the culture system and possible factors involved in growth and differentiation of epithelial cells are discussed.

Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules

Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1–10 μg/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1α and IL-1β, and IL-6, IL-8 and TNF-α as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria.

Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-laminin receptor

The incorporation of a radioactive precursor 3H-glucosamine in glycoconjugates, essentially glycosaminoglycans (GAG) was evaluated in the culture medium and cell fraction of human skin fibroblasts. Using increasing passage numbers, we could estimate the effect of in vitro aging on these biosynthetic activities. The incorporation in different free (hyaluronan) and protein bound (proteoglycans) GAGs was evaluated after specific enzymatic digestion. Most newly synthesized GAGs were excreted in the extracellular medium. Incorporation of the tracer in hyaluronan, the major biosynthetic product, increased with passage number but its titratable concentration decreased with in vitro aging, suggesting a rapid post-synthetic degradation. The proportion of chondroitin sulfates 4 (A) and 6 (C) and heparan sulfate decreased and that of dermatan sulfate increased with increasing passage number. We explored the modulation of these biosynthetic activities by the elastin laminin receptor. Using agonists (elastin peptides) and an antagonist (melibiose) of the receptor, their action on GAG biosynthesis was evaluated. Both elastin peptides and melibiose increased incorporation of the tracer in GAGs, but only melibiose inhibited post-synthetic degradation of hyaluronan, therefore increasing its concentration. The effect of passage number on the receptor mediated modulations was also investigated.

Mammary fibroblast-derived hepatocyte growth factor stimulates growth and morphogenesis of mouse mammary tumor cells in primary culture.

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three‐dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth‐stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth‐stimulating activity present in the mammary fibroblast‐conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast‐derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.

A novel 4-methylumbelliferyl-beta-D-xyloside derivative, sulfate-O-3-xylosylbeta1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis.

Human skin fibroblasts were incubated in the presence of 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU). The culture medium was recovered and Xyl-MU derivatives which were initiated by the Xyl-MU acting as a primer were purified. As a result, a novel Xyl-MU derivative was isolated, in addition to previously reported Xyl-MU derivatives such as glycosaminoglycan-MU, Gal-Gal-Xyl-MU, Gal-Xyl-MU, SA-Gal-Xyl-MU, Xyl-Xyl-MU, GlcA-Xyl-MU, and sulfate-GlcA-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, ion-spray mass spectrometric analysis, and Smith degradation. The results indicated that it was sulfate- O -3-Xyl-MU. When Xyl-MU was incubated with [35S]PAPS using a homogenate prepared from the same cultured skin fibroblasts, [35S]sulfate- O -3-Xyl-MU was produced. Moreover, when Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was transferred to Xyl-MU, but when sulfate- O -3-Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was not transferred. These results indicate that chain elongation from Xyl-MU is inhibited by sulfation of Xyl-MU, and that Xyl-MU sulfation is involved in the control of Xyl-MU-initiated glycosaminoglycan biosynthesis.