Human Embryonic Skin Fibroblasts Research Articles

Protective Mechanism of Rosa roxburghii Tratt Fermentation Broth against Ultraviolet-A-Induced Photoaging of Human Embryonic Skin Fibroblasts.

This study takes the fruit of Rosa roxburghii Tratt (RRT) as a fermentation substrate and carries out a quantitative visual analysis of the domestic and foreign literature on screenings of five different lactic acid bacteria to obtain a fermentation broth. Systemic anti-photoaging effects are analyzed at the biochemical, cellular, and molecular biological levels. DPPH and ABTS free radical scavenging activities are used to verify the antioxidant capacity of the RRT fruit fermentation broth in vitro. Human embryonic skin fibroblasts (HESs) are used to establish a UVA damage model, and the antioxidant capacity of the RRT fruit fermentation broth is verified in terms of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. RT-qPCR and ELISA are used to detect the expression of TGF-β/Smad, MMPs, and the MAPK/AP-1 and Nrf2/Keap-1 signaling pathways in order to explore the anti-oxidation and anti-photoaging effects of the RRT fruit fermentation broth by regulating different signaling pathways. The results show that an RRT fruit fermentation broth can effectively protect cells from oxidative stress caused by UVA and has significant anti-photoaging effects, with the co-cultured Lactobacillus Yogurt Starter LYS-20297 having the highest overall effect.

Effects of Photobiomodulation on High Glucose Induced Oxidative Stress in Human Embryonic Skin Fibroblasts

In this study, we explored the effects of photobiomodulation (PBM) on the functional damage of human embryonic skin fibroblasts (CCC-ESFs) in a high glucose environment, as well as the effects of PBM on tissue repair, to provide a theoretical basis for application and promotion of dual-wavelength laser therapy in clinical practices. CCC-ESFs were cultured in vitro for 2 d in a high glucose medium, and then irradiated with a 635/808 nm dual-wavelength semiconductor laser at a dose of 20 mW/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , 0-24 J/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> . Cell proliferation, reactive oxygen species (ROS) production, antioxidant enzyme activity (CAT, SOD), lipid oxidization capacity (MDA), cell growth factors, and the expression of matrix metalloproteinases (MMPs) and matrix metalloproteinase inhibitors (TIMPs) were assessed. We found that PBM can enhance the proliferation of fibroblasts, improve the inhibitory effects of the high glucose environment on cell proliferation, increase intracellular ROS production, enhance the activity of antioxidant enzymes, and stimulate cytokine secretion. Furthermore, PBM was found to inhibit the expression of MMPs, increase the expression of TIMPs, reverse the overexpression of MMPs/TIMPs caused by high glucose, and promote wound healing.

Inhibition of sphingosine kinase2 attenuates hypertrophic scar formation via upregulation of Smad7 in human hypertrophic scar fibroblasts.

The aims of the present study were to investigate the role of sphingosine kinase 2 (Sphk2) in hypertrophic scar (HS) formation and its underlying mechanisms. The expression levels of Sphk2 and Smad7 in HS tissues and healthy skin tissues of patients undergoing plastic surgery were determined using immunohistochemical staining. Subsequently, the expression levels of Sphk2 and collagen I in human embryonic skin fibroblasts (control) and human HS fibroblasts (HSF) were detected using western blot analysis and immunofluorescence assay, respectively. Following Sphk2 silencing, Smad7 overexpression or both Sphk2 and Smad7 silencing, HSF proliferative ability was assessed using Cell Counting Kit-8 assay and proliferation-associated proteins were evaluated using western blot analysis. In addition, the level of apoptosis in HSF was assessed using flow cytometry and expression levels of apoptotic-associated proteins were determined using western blotting. Furthermore, the expression levels of collagen I and proteins in the TGF-β1/Smad signaling pathway were detected using western blot analysis. The results indicated that the expression of Sphk2 was significantly increased, while Smad7 expression was decreased in HS tissue. Moreover, the upregulation of Sphk2 and collagen I expression levels was identified in HSF. The present results also indicated that Sphk2 silencing or Smad7 overexpression inhibited proliferation, but promoted apoptosis of HSF, coupled with changes in the expression levels of proliferation-associated proteins, with an increase in p21 and a decrease in cyclin D1 expression levels, and apoptosis-associated proteins, with an increase in Bax and cleaved caspase-3, and a decrease in Bcl-2, which were reversed following transfection with both Sphk2 and Smad7 using small interfering RNA in HSF. In addition, the expression levels of transforming growth factor-β1, phosphorylated (p)-Smad2, p-Smad3 and collagen I were reduced following Sphk2 silencing or Smad7 overexpression, which were abolished by silencing both Sphk2 and Smad7. Collectively, the present results indicated that inhibition of Sphk2 attenuated HS formation via upregulation of Smad7 expression, thus Sphk2 may serve as a potential therapeutic target for the treatment of HS.

The transdermal performance, pharmacokinetics, and anti-inflammatory pharmacodynamics evaluation of harmine-loaded ethosomes

Harmine (HAR) is a β-carboline alkaloid with anti-inflammatory and antipruritic effect. However, the low bioavailability and side effects of HAR severely limited its clinical application. The main objective of this study was to develop harmine-loaded ethosomes (HLE) drug delivery system for topical application to treat inflammation. HLE were obtained by ethanol injection method and characterized. The morphology of HLE was evaluated by transmission electron microscopy (TEM). HLE exhibited a good biocompatibility with human embryonic skin fibroblasts and rat skin. The in vitro skin penetration studies showed that HLE had the greatest skin deposition than harmine-loaded liposomes (HLL) and harmine solution (HS). In vivo pharmacokinetic study demonstrated that AUC(0–∞) and Cmax of HLE in subcutaneous tissues were much higher than that of in blood. Moreover, for convenience of fixing on skin, HLE were mixed with gel. HLE gel significantly inhibited the overexpression of inflammation cytokines prostaglandin E2, interleuking (IL)-1β, nitric oxide, and tumor necrosis factor-alpha (TNF-α) in the inflammation model of rat paw edema compared with HS gel. In short, HLE was promising formulation for topical delivery in treatment of inflammatory diseases.

Inhibitory Effect of Metalloproteinase Inhibitors on Skin Cell Inflammation Induced by Jellyfish Nemopilema nomurai Nematocyst Venom.

Jellyfish envenomations result in extensive dermatological symptoms, clinically named as jellyfish dermatitis, which can seriously affect the daily activities and physical health of people. Inflammatory response accompanies the whole process of jellyfish dermatitis and the complexity of jellyfish venom components makes it difficult to treat jellyfish dermatitis symptoms effectively. Moreover, inhibiting inflammation is essential for the treatment of jellyfish stings and exploring the main components of jellyfish venom that cause inflammation is an urgent research area. In this study, the inhibitory effects of matrix metalloproteinase (MMP) inhibitors for venom-induced inflammation were explored at a cellular level. The expression of the three inflammatory factors, IL-6, TNF-α and MCP-1 in two skin cell lines, human keratinocyte cells (HaCaT) and human embryonic skin fibroblasts cells (CCC-ESF-1), at the cellular level, after treatment with the inhibitors of jellyfish Nemopilema nomurai (N. nomurai) nematocyst venom (NnNV-I), were determined. The results showed that inhibitors of MMP can significantly reduce the toxic effects of jellyfish Nemopilema nomurai nematocyst venom (NnNV) to skin cells. The expression levels of the three inflammatory factors IL-6, MCP-1, and TNF-α in the cells were also significantly decreased, indicating that MMPs in jellyfish venom are probably vital factors leading to jellyfish dermatitis. This study is beneficial in the prevention and treatment of jellyfish stings.

Polycomb group proteins: Novel molecules associated with ultraviolet A-induced photoaging of human skin.

Epigenetic repressor polycomb group (PcG) proteins are thought to serve a role in a number of cellular processes, including carcinogenesis, senescence, apoptosis and DNA repair. In the present study, long-wave ultraviolet A (UVA) was used to irradiate human skin fibroblasts (HSFs) and embryonic skin fibroblasts (ESFs) in order to simulate photoaging of the skin. The results of cell proliferation, apoptosis, hyaluronic acid (HA) content and reverse transcription-quantitative polymerase chain reaction assays revealed that the expression levels of genes encoding key PcG proteins (BMI-1 and EZH2) were altered. In addition, the expression levels of these genes were associated with the expression of enzymes that regulate HA synthesis. Furthermore, the expression levels of PcG proteins differed between HSFs and ESFs, suggesting that PcG proteins serve a role in altering HA synthesis during the UVA-induced fibroblast aging process. This signaling pathway may represent a novel molecular mechanism regulating the photoaging of the skin. The findings of the present study provide important insights into the underlying mechanisms of photoaging of the human skin. Further studies are required to clarify the molecular mechanisms underling skin aging and to identify targets for the clinical treatment of photoaging.

Effects of genistein on the mRNA expressions of collagen, matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase in human embryonic skin fibroblasts

Objective To investigate the effects of genistein on the mRNA expressions of collagen (Col), matrix metalloproteinase (MMP ) and tissue inhibitor of matrix metalloproteinase (TIMP) in human embryonic skin fibroblasts (CCC-ESF-1). Methods The cultured CCC-ESF-1cells were divided into a black control group, an estradiol group and genistein groups of different doses. The mRNA expressions of ColⅠ , Col Ⅲ, MMP-1, TIMP-1 and TIMP-2 were detected by RT-PCR. Results Compared with the black group, estradiol and medium dose of genistein (0.451 ± 0.037, 0.446 ± 0.047 vs. 0.385 ± 0.061, all P<0.05) could promote the proliferation of the CCC-ESF-1 cells, estradiol and medium dose of genistein could up-regulate the mRNA expressions of ColⅠ (0.960 ± 0.012, 0.929 ± 0.015 vs. 0.812 ± 0.014, all P<0.01), Col Ⅲ (0.892 ± 0.009, 0.824 ± 0.022 vs. 0.768 ± 0.025, all P<0.01), TIMP-1 (0.841 ± 0.023, 0.838 ± 0.053 vs. 0.751 ± 0.027, all P<0.01) and TIMP-2 (0.456 ± 0.017, 0.448 ± 0.036 vs. 0.381 ± 0.029, all P<0.01), and down-regulate MMP-1 mRNA expression (0.398 ± 0.043, 0.402 ± 0.044 vs. 0.525 ± 0.006, all P<0.01). Conclusions Genistein could promote the proliferation of the CCC-ESF-1 cells, and that may be related with up-regulating the mRNA expressions of ColⅠ,Col Ⅲ, MMP-1, TIMP-1and down-regulating MMP-1 mRNA expression. Key words: Fibroblasts; Genistein; Collagen; Matrix metalloproteinase 1; Matrix metalloproteinase inhibitors

Evaluation of transdermal salidroside delivery using niosomes via in vitro cellular uptake

Span 40-based niosomes were employed as nanocarriers to improve cutaneous absorption of salidroside. The niosomal formulation with a molar proportion of Span 40 to cholesterol of 4:3 showed the highest transdermal flux and skin deposition of salidroside. The transdermal flux of the 4:3 niosomal formulation was significantly greater than that of the aqueous solution. Salidroside-loaded niosomes showed good biocompatibility with skin tissue, human epidermal immortal keratinocytes (HaCaT), and human embryonic skin fibroblasts (CCC-ESF). The fluorescence intensity of HaCaT cells after uptake of coumarin 6-labeled niosomes was similar to that observed after uptake of the aqueous suspension. The fluorescence intensity of CCC-ESF cells was greater than that of the aqueous suspension after incubation for 10min, but was not significantly different after 60min. Further investigation revealed that internalization of niosomes by HaCaT cells may be achieved through pinocytotic vesicles and macropinocytosis, which consumes energy, rather than via lysosomes. In CCC-ESF cells, pinocytotic vesicles and lysosomes were both important mediators of endocytosis. The niosome formulations reported here could improve the dermal and transdermal salidroside delivery, and the in vitro cell uptake evaluation results serve as a basis for further research into the mechanisms through which niosomes enhance drug permeability.

Preparation and evaluation of microemulsion-based transdermal delivery of total flavone of rhizoma arisaematis.

The aims of the present study were to investigate the skin permeation and cellular uptake of a microemulsion (ME) containing total flavone of rhizoma arisaematis (TFRA), and to evaluate its effects on skin structure. Pseudo-ternary phase diagrams were constructed to evaluate ME regions with various surfactants and cosurfactants. Eight formulations of oil-in-water MEs were selected as vehicles, and in vitro skin-permeation experiments were performed to optimize the ME formulation and to evaluate its permeability, in comparison to that of an aqueous suspension. Laser scanning confocal microscopy and fluorescent-activated cell sorting were used to explore the cellular uptake of rhodamine 110-labeled ME in human epidermal keratinocytes (HaCaT) and human embryonic skin fibroblasts (CCC-ESF-1). The structure of stratum corneum treated with ME was observed using a scanning electron microscope. Furthermore, skin irritation was tested to evaluate the safety of ME. ME formulated with 4% ethyl oleate (weight/weight), 18% Cremophor EL® (weight/weight), and 18% Transcutol® P, with 1% Azone to enhance permeation, showed good skin permeability. ME-associated transdermal fluxes of schaftoside and isoschaftoside, two major effective constituents of TFRA, were 3.72-fold and 5.92-fold higher, respectively, than those achieved using aqueous suspensions. In contrast, in vitro studies revealed that uptake by HaCaT and CCC-ESF-1 cells was lower with ME than with an aqueous suspension. Stratum corneum loosening and shedding was observed in nude mouse skin treated with ME, although ME produced no observable skin irritation in rabbits. These findings indicated that ME enhanced transdermal TFRA delivery effectively and showed good biocompatibility with skin tissue.

Comparison of ethosomes and liposomes for skin delivery of psoralen for psoriasis therapy

Recent reports have indicated that psoriasis may be caused by malfunctioning dermal immune cells, and psoralen ultraviolet A (PUVA) is an effective treatment for this chronic disease. However, conventional topical formulations achieve poor drug delivery across patches of psoriasis to their target sites. The present study describes the development of a novel psoralen transdermal delivery system employing ethosomes, flexible vesicles that can penetrate the stratum corneum and target deep skin layers. An in vitro skin permeation study showed that the permeability of psoralen-loaded ethosomes was superior to that of liposomes. Using ethosomes, psoralen transdermal flux and skin deposition were 38.89±0.32μg/cm2/h and 3.87±1.74μg/cm2, respectively, 3.50 and 2.15 times those achieved using liposomes, respectively. The ethosomes and liposomes were found to be safe following daily application to rat skin in vivo, for 7 days. The ethosomes showed better biocompatibility with human embryonic skin fibroblasts than did an equivalent ethanol solution, indicating that the phosphatidylcholine present in ethosome vesicles improved their biocompatibility. These findings indicated that ethosomes could potentially improve the dermal and transdermal delivery of psoralen and possibly of other drugs requiring deep skin delivery.

Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Resistance to chemotherapy is one of the principal causes of cancer mortality and is generally considered a late event in tumor progression. Although cellular models of drug resistance have been useful in identifying the molecules responsible for conferring drug resistance, most of these cellular models are derived from cell lines isolated from patients at a late stage in cancer progression. To ask at which stage in the tumorigenic progression does the cell gain the ability to acquire drug resistance, we generated a series of pre-tumorigenic and tumorigenic cells from human embryonic skin fibroblasts by introducing, sequentially, the catalytic subunit of telomerase, SV40 large T and small T oncoproteins, and an oncogenic form of ras. We show that the ability to acquire multidrug resistance (MDR) can arise before the malignant transformation stage. The minimal set of changes necessary to obtain pre-tumorigenic drug-resistant cells is expression of telomerase and inactivation of p53 and pRb. Thus, the pathways inactivated during tumorigenesis also confer the ability to acquire drug resistance. Microarray and functional studies of drug-resistant pre-tumorigenic cells indicate that the drug efflux pump P-glycoprotein is responsible for the MDR phenotype in this pre-tumorigenic cell model.

The collagenase inhibitor from human polymorphonuclear leukocytes. Isolation, purification and characterisation.

A new collagenase inhibitor of latent human polymorphonuclear collagenase, initially reported by Macartney and Tschesche [Hoppe-Seyler's Z. Physiol. Chem. 361, 298–299 (1980) and FEBS Lett. 119, 327–332 (1980)], has been purified to apparent homogeneity. We described several methods of purification of this inhibitor, but the fastest and most reproducible method is by passage of purified latent human polymorphonuclear leukocyte collagenase on activated thiol-Sepharose 4-B (preceding paper in this journal). Active collagenase is eluted from the column and the collagenase inhibitor is retarded. Elution of the inhibitor is achieved by eluting the column with thiol compounds in the equilibration buffer. Further purification of the inhibitor is obtained by gel filtration on Sephacryl S-300 and ion-exchange chromatography on DEAE-Sephacel. The inhibitor exhibits a strict specificity for mammalian collagenases. It inhibits collagenases isolated from human synovial fluid, human normal and diabetic skin fibroblasts and human embryonic skin fibroblasts. The inactive complexes are all subject to activation by several disulfide compounds, such as oxidised glutathione by the thiol/disulfide interchange reaction already described. The purified inhibitor was shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (10% gel) to be homogeneous and displayed an apparent molecular weight of 24500. Titration of the inhibitor with Ellmans reagent revealed that it contains one free sulfhydryl group, a prerequisite for its inhibitory activity. The inhibitor inhibits human polymorphonuclear leukocyte collagenase in a strict 1:1 stoichiometric reaction. The amino acid composition of the complex formed from the inhibitor and active enzyme reproduces that of the latent collagenase thus confirming that the latent enzyme is a 1:1 complex. The inhibitor is an acidic protein having an isoelectric point at pH 5.5, which would be expected from the amino acid analysis. The composition reveals a glycoprotein of about 240 residues containing a carbohydrate moiety composed of N-acetylglucosamine, mannose, galactose and glucose. The amino acid composition of the inhibitor is not identical to that of β1-anticollagenase from human plasma.

In vitro cytotoxicity testing with fluorescence-based assays in cultured human lung and dermal cells.

An in vitro study using human cultured cells was conducted to determine the reliability of fluorescence-based cell viability indicators with traditional in vitro cytotoxicity testing methods. Human lung epithelial carcinoma (A549) cells, and human embryonic skin (WS1) and lung (HFLI) fibroblasts were studied in culture to evaluate their potential to screen for cytotoxicity and to compare to previous protocols conducted in our laboratory. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and fluorescent-labeled probes were used to assess toxicity. Eight chemicals, including mercuric chloride, copper sulfate, sodium fluoride, thioridazine HCl, paraquat, amitriptyline-HCl, verapamil-HCl and chloroquine sulfate, were tested with each cell line using calcein-AM and Sytox. The data suggest that fluorescent probes are sensitive indicators of cytotoxicity and contribute to understanding the mechanisms for each chemical. In combination with previously published reports, the similarity of results among cell lines may be explained by the origin of the cell lines rather than by the diversity of the methods and indicators employed.

Human skin fibroblasts induce anchorage‐independent growth of HPV‐16‐DNA‐immortalized cervical epithelial cells

Immortalized non-malignant cells do not grow in soft agarose. We found, however, that HPV-16-DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) formed colonies when co-cultured with human embryonic skin fibroblasts. This appeared to be mediated by diffusible growth factors, and direct cell-cell contacts were not required. HCE16/3 cells from colonies remained unable to grow alone in soft agarose like the parental HCE16/3 cells, indicating that only transient phenotypic changes without genetic alterations had occurred in the co-cultures. In a modified soft-agarose assay, HCE16/3 cells grown as an underlying monolayer did not produce enough (transforming) growth factors which could confer an anchorage-independent phenotype to non-transformed fibroblasts. Lethally irradiated fibroblasts were able to stimulate DNA synthesis and proliferation of HCE16/3 cells. Northern-blot analysis showed that HPV-16-DNA expression of the immortalized cells was not altered in the presence of fibroblasts. The expression of the IL-I gene in the immortalized cells, however, was augmented by the co-cultured fibroblasts compared with immortalized cells alone. In organotypic collagen raft co-cultures, in which epithelial cells were grown on top of a collagen gel containing fibroblasts, HCE16/3 cells appeared to be chemotactic to fibroblasts and fibroblasts stimulated the growth of the immortalized epithelial cells. Our results suggest that fibroblasts may contribute to human epithelial carcinogenesis in vivo.

Keratinocyte growth factor is a bifunctional regulator of HPV16 DNA-immortalized cervical epithelial cells.

Various factors are known to regulate cell growth and differentiation in epithelial-mesenchymal interactions. Keratinocyte growth factor (KGF), an epithelial-specific cytokine produced by dermal fibroblasts and other mesenchymal cells, appears to affect growth, migration, and differentiation in epithelial-mesenchymal interactions. We have previously shown that human embryonic skin fibroblasts induce anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in cocultures of HCE16/3 cells and fibroblasts. Here we report that KGF may be a major factor influencing growth and behavior of HCE16/3 cells in the coculture system. KGF stimulated both DNA synthesis and proliferation of normal human cervical epithelial (HCE) cells and HCE16/3 cells and the increase was stronger in HCE16/3 cells than in HCE cells. SiHa cells, a cervical carcinoma cell line with integrated HPV16 DNA, did not respond to the KGF mitogen signal. KGF receptor (KGFR) studies suggested that the different responses to the KGF mitogen signal may be correlated with KGFR. In addition, KGF alone was able to induce anchorage-independent growth of HCE16/3 cells, suggesting a potential role for KGF in the transformation process of epithelial cells. However, the transcription of HPV16 early genes was suppressed by KGF in the immortalized HCE16/3 cells, and this appeared to be due to transcriptional repression rather than a posttranscriptional process according to nuclear run-on analysis. In contrast, viral gene expression was not affected by KGF in SiHa cells. Our results suggest that KGF is a bifunctional growth factor in the HPV-immortalized cells, a positive regulator of cell growth and negative regulator of HPV16 early gene expression.

Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts

(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl β- d-xylopyranoside (and [ 3H]galactose) or 125I-labelled p-hydroxyphenyl β- d-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous β- d-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.

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

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]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-beta 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-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl 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 beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.

Modulation of phospholipase A2 activity in human fibroblasts

1. Human embryonic skin fibroblasts (HSF) incubated overnight with either human recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta) released large amounts of prostaglandin E2 (PGE2). 2. rIL-1 beta, bradykinin (Bk) and arachidonic acid (AA) significantly stimulated PGE2 release from HSF incubated overnight in the presence of either interleukin. 3. Hydrocortisone inhibited the PGE2 release induced by rIL-1 beta and Bk, but not by AA. 4. The steroid inhibitory effect was reversed by actinomycin D as well as by an anti-lipocortin monoclonal antibody. 5. The results suggest that in HSF, rIL-1 beta is able to stimulate both cyclo-oxygenase and phospholipase A2 (PLA2) activity. 6. The stimulation of PLA2 activity by rIL-1 beta is inhibited by hydrocortisone, probably via induction of lipocortin-like proteins.

?-L-fucosidase secretion by human embryonic skin fibroblasts

?-L-fucosidase secretion by human embryonic skin fibroblasts

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.