Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.
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ABSTRACT:
SUBMITTER: Heldin P
PROVIDER: S-EPMC1138453 | biostudies-other | 1989 Mar
REPOSITORIES: biostudies-other
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Project description:The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappaB (nuclear factor kappaB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta1 (transforming growth factor-beta1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.
| S-EPMC1868797 | biostudies-literature
Project description:Hyaluronans (HAs) are glycosaminoglycans produced in the bodies of Anguilliform and Elopiform leptocephali, and play a role in metabolic energy. In mammals, HA synthesis-promoting substances (HASPS) up-regulate the expression of HA synthase (HAS) and increase the amount of HA in the body. In this study, Japanese eel leptocephali were fed a HASPS containing diet. We analyzed HAS1s and HAS2 expression, HA content, and their influence on growth. HASPS extracted from Grifola frondosa promoted HAS1s and HAS2 mRNA and HA content. Other than mammals, these results are first reported in vertebrate. Moreover, HASPS extracted from G. frondosa promoted leptocephalus growth. The relationship between growth and HA in the leptocephali is not yet clear. However, based on our results we hypothesize that HA is involved in the storage of energy, which is metabolized to sugars when needed for metabolic energy.
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Project description:When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase-sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms.
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Project description:The aim of this study was to identify, in cultured human cervical fibroblasts, the mechanisms by which interleukin (IL)-1beta induces the synthesis of glycosaminoglycans (GAG) and to explore the putative role of prostaglandin E(2) (PGE(2)) in this process. Exposure of the cells for 24 h to IL-1beta induced a significant (P < 0.05) dose-dependent increase in GAG synthesis. IL-1beta (1 ng/ml) induced the expression of cyclooxygenase-2 (COX-2) protein 6 h after treatment, accompanied by a 7.5-fold increase in PGE(2) production. We confirmed that NS398, a selective COX-2 inhibitor, dose-dependently blocked PGE(2) augmentation following IL-1beta treatment. AH23848, the selective EP(4) receptor antagonist, completely abolished IL-1beta-induced GAG synthesis, whereas AH6809, an EP(2) receptor antagonist, had no effect on the stimulatory effects of IL-1beta. Furthermore, we demonstrated that 6 h exposure to IL-1beta induced a notable increase in EP(4) receptor mRNA expression and a decrease in EP(1) receptor mRNA but had no effect on the expression of EP(2) and EP(3) receptor transcripts. In conclusion, these findings indicate that IL-1beta not only induced GAG synthesis by increasing COX-2 protein expression and the subsequent PGE(2) production but also enhanced the responsiveness of cervical fibroblasts to PGE(2) by selectively up-regulating EP(4) receptor mRNA expression. These results suggest that PGE(2) may regulate human cervical ripening in an autocrine/paracrine manner via EP(4) receptors.
| S-EPMC4710740 | biostudies-literature
Project description:Background and objectiveThe potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration.Material and methodsMetabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing-related gene expression and activation of signaling kinases were analyzed by qRT-PCR and immunoblotting, respectively.ResultsThe investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor-β3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro-proliferative, pro-migratory, and pro-inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro-inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts.ConclusionBoth investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.
| S-EPMC6586051 | biostudies-literature
Project description:A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
| S-EPMC1162088 | biostudies-other
Project description:Hyaluronan (HA) is a glycosaminoglycan located in the interstitial space which is essential for both structural and cell regulatory functions in connective tissue. We have previously shown that HA synthesis is up-regulated in a rat model of experimental cardiac hypertrophy and that cardiac tissue utilizes two different HA synthases in the hypertrophic process. Cardiomyocytes and fibroblasts are two major cell types in heart tissue. The fibroblasts are known to produce HA, but it has been unclear if cardiomyocytes share the same feature, and whether or not the different HA synthases are activated in the different cell types.This study shows, for the first time that cardiomyocytes can produce HA. Cardiomyocytes (HL-1) and fibroblasts (NIH 3T3) were cultivated in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2. HA concentration was quantified by ELISA, and the size of HA was estimated using dynamic light scattering. Cardiomyocytes synthesized HA but only when stimulated by PDGF-BB, whereas fibroblasts synthesized HA without addition of growth factors as well as when stimulated by any of the three growth factors. When fibroblasts were stimulated by the growth factors, reverse dose dependence was observed, where the highest dose induced the least amount of HA. With the exception of TGFB2, a trend of reverse dose dependence of HA size was also observed.Co-cultivation of cardiomyocytes and fibroblasts (80%/20%) increased HA concentration far more that can be explained by HA synthesis by the two cell types separately, revealing a crosstalk between cardiomyocytes and fibroblasts that induces HA synthesis. We conclude that dynamic changes of the myocardium, such as in cardiac hypertrophy, do not depend on the cardiomyocyte alone, but are achieved when both cardiomyocytes and fibroblasts are present.
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Project description:Magnesium inadequacy affects more than half of the U.S. population and is associated with increased risk for many age-related diseases, yet the underlying mechanisms are unknown. Altered cellular physiology has been demonstrated after acute exposure to severe magnesium deficiency, but few reports have addressed the consequences of long-term exposure to moderate magnesium deficiency in human cells. Therefore, IMR-90 human fibroblasts were continuously cultured in magnesium-deficient conditions to determine the long-term effects on the cells. These fibroblasts did not demonstrate differences in cellular viability or plating efficiency but did exhibit a decreased replicative lifespan in populations cultured in magnesium-deficient compared with standard media conditions, both at ambient (20% O(2)) and physiological (5% O(2)) oxygen tension. The growth rates for immortalized IMR-90 fibroblasts were not affected under the same conditions. IMR-90 fibroblast populations cultured in magnesium-deficient conditions had increased senescence-associated beta-galactosidase activity and increased p16(INK4a) and p21(WAF1) protein expression compared with cultures from standard media conditions. Telomere attrition was also accelerated in cell populations from magnesium-deficient cultures. Thus, the long-term consequence of inadequate magnesium availability in human fibroblast cultures was accelerated cellular senescence, which may be a mechanism through which chronic magnesium inadequacy could promote or exacerbate age-related disease.
| S-EPMC2311331 | biostudies-literature
Project description:BackgroundAlthough deubiquitinating enzymes (DUBs) such as CYLD, A20 and OTULIN are expressed in multiple tissues and thought to be linked with inflammatory diseases, their expression in periodontal tissues remains to be determined. This research was designed to assess the expression of CYLD, A20 and OTULIN in human gingiva, and to evaluate the regulation of these DUBs in human gingival fibroblasts (HGFs) upon different stimuli.MethodsImmunohistochemistry assay was conducted to determine the expression of CYLD, A20 and OTULIN in human gingiva. Immunofluorescence assay was employed to observe the protein expression of CYLD, A20 and OTULIN in HGFs. RT-PCR and western blots were carried out to assess gene and protein expression changes of these DUBs in HGFs upon LPS or TNF-α.ResultsCYLD, A20 and OTULIN were found to be expressed in human gingiva and HGFs. The expression of CYLD, A20 and OTULIN was lower in the inflamed gingival tissue samples compared with the healthy gingival tissue samples. Further, the expression of CYLD, A20 and OTULIN in HGFs exhibited distinct regulation by different stimuli. TNF-α treatment markedly increased NF-κB activation in HGFs CONCLUSIONS: Our findings suggest that CYLD, A20 and OTULIN might play a role in the progression of periodontitis.
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Project description:Background and purposeWorldwide, oesophageal cancer is the eighth most common cancer and has a very poor survival rate. In order to identify new tolerable treatment options for oesophageal squamous cell carcinoma (ESCC), erlotinib was tested with moderate efficacy in phase I and II studies. As 4-methylumbelliferone (4-MU), an hyaluronan (HA) synthesis inhibitor showed anti-cancer effects in vitro, and in ESCC xenograft tumours, we investigated whether the anti-cancer effects of erlotinib could be augmented by combining it with 4-MU.Experimental approachESCC cell lines were treated with erlotinib or gefitinib (1 μmol·L-1 ) and 4-MU (300 μmol·L-1 ), and the cell count, cell cycle progression and migration were determined as compared to the single agents and the solvent-control.Key resultsThe combination of erlotinib and 4-MU synergistically inhibited the proliferation of ESCC cell lines. Furthermore, the migration speed of ESCC cell line KYSE-410 in gap closure assays was significantly reduced by the combination of erlotinib and 4-MU. Decreased ERK phosphorylation could explain the anti-proliferative and anti-migratory effects in the combined treatment group. Finally, the combination was additionally able to decrease the growth of multicellular tumour spheroids, a three-dimensional cell culture model that was associated with sustained inhibition of ERK1/2 phosphorylation.Conclusions and implicationsThe combination of 4-MU and erlotinib showed promising anti-cancer efficacies in the ESCC cell lines.
| S-EPMC4562515 | biostudies-literature