Effect of fibroblast growth factor-1 on the expression of early growth response-1 in human periodontal ligament cells.

BACKGROUND AND OBJECTIVES: Early growth response-1 is a nuclear transcription factor implicated in regulating cell proliferation. Fibroblast growth factor-1 is the prototypic fibroblast growth factor involved in the proliferation and differentiation of various cell types. Expression of early growth response-1 induced by fibroblast growth factor-1 thus may be very important for cell growth, during both development and wound healing in oral tissue. However, little is known about the expression and kinetics of early growth response-1 in fibroblast growth factor-1-stimulated oral cells. The aim of this study was to investigate the effects of fibroblast growth factor-1 on the expression of early growth response-1 in human periodontal ligament cells. MATERIAL AND METHODS: Periodontal ligament cells were cultured in medium containing 1, 10 or 100 ng/mL of fibroblast growth factor-1 for 45 min or with 10 ng/mL of fibroblast growth factor-1 for 15, 30, 45, 60 or 120 min. The proliferation of periodontal ligament cells was evaluated by measuring 5-bromo-2'-deoxyuridine incorporation. The expression of early growth response-1 mRNA and protein, and the localization of early growth response-1 protein, were examined by western blotting, northern blotting and immunocytostaining. RESULTS: 5-Bromo-2'-deoxyuridine incorporation correlated directly with increases in fibroblast growth factor-1 concentration, and 5-bromo-2'-deoxyuridine incorporation peaked 45 min after starting treatment. Early growth response-1 protein was expressed in response to a concentration of fibroblast growth factor-1 as low as 1 ng. Peak expression of early growth response-1 mRNA was observed at 15 min and that of early growth response-1 protein at 60 min. The 140-kDa early growth response-1 protein was not detected in the nuclear fraction, and the peak expression of the 80-kDa early growth response-1 protein occurred at 60 min. Early growth response-1 localized in or around the nucleus at 30 min. CONCLUSION: These results show that a concentration of fibroblast growth factor-1 as low as 1 ng induces the expression of early growth response-1 protein, and that the 80-kDa early growth response-1 protein functions in the nucleus of periodontal ligament cells treated with fibroblast growth factor-1.

Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways.

The cysteine-rich protein 61 (Cyr61) is a signaling molecule with functions in cell migration, adhesion, and proliferation. This protein is encoded by an immediate early gene whose expression is mainly induced by serum growth factors. Here we show that Cyr61 mRNA levels increase sharply in response to cyclic mechanical stretch applied to cultured bladder smooth muscle cells. Stretch-induced changes of Cyr61 transcripts were transient and accompanied by an increase of the encoded protein that localized mainly to the cytoplasm and nucleus of the cells. With the use of pharmacological agents that interfere with known signaling pathways, we show that transduction mechanisms involving protein kinase C and phosphatidylinositol 3-kinase activation partly blocked stretch-induced Cyr61 gene expression. Selective inhibition of Rho kinase pathways altered this stretch effect as well. Meanwhile, using inhibitors of the actin cytoskeleton, we show that Cyr61 gene expression is sensitive to mechanisms that sense actin dynamics. These results establish the regulation of Cyr61 gene by mechanical stretch and provide clues to the key signaling molecules involved in this process.

Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.

Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.

Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappaB transcription factor.

In this study, we used the platelet-activating factor (PAF) receptor gene as a model of a mechano-sensitive gene to investigate how mechanical stimuli regulate gene expression and cell function. We utilized a culture system of pulmonary artery smooth muscle cells and a well-defined in vitro mechanical device that imparts an equibiaxial strain repeatedly to cells attached to an elastomeric membrane. Northern blot and immunohistochemical analyses revealed increased PAF receptor expression at both the mRNA and protein levels after 1 h exposure of the cells to a 5% strain at a frequency of 1 Hz. To investigate the mechanism of activation of this gene by stretch, we performed transfection experiments with a luciferase reporter gene linked to segments of the 5' flanking region of the receptor gene promoter. Expression of the transfected reporter gene bearing a 1.1-kb fragment of the promoter was enhanced in mechanically stretched cells indicating a direct effect on transcriptional activity. When truncated to leave the nucleotides between -610 to +27, the promoter-reporter construct lost stretch inducibility suggesting that the region between -1099 and -610 was required for stretch responsiveness. This region contains four copies of NF-kappaB binding sites. These elements are in close proximity to one another and can form a complex with nuclear proteins derived from stretched cells as demonstrated by gel mobility shift assay. Moreover, in experiments using cycloheximide, we found that de novo protein synthesis was not necessary for the induction of the PAF receptor gene expression by mechanical stretch. Conversely, preincubation of the cells with protein kinase C inhibitors suppressed mechanical stretch-induced PAF receptor gene expression at the mRNA levels and abrogated upstream events of NF-kappaB activation in the cytoplasm. These data strongly suggest that stretch-induced PAF receptor gene expression is mediated by NF-kappaB binding to the PAF receptor gene promoter and that protein kinase C activation is among the molecular features of NF-kappaB activation and translocation into the nucleus in mechanically stretched cells.

Influence of transforming growth factor beta1 (TGF-beta1) on the behaviour of porcine thyroid epithelial cells in primary culture through thrombospondin-1 synthesis.

Transforming growth factor beta1 (TGF-beta1) is a secreted polypeptide that is thought to play a major role in the regulation of folliculogenesis and differentiation of thyroid cells. On porcine thyroid follicular cells cultured on plastic substratum, TGF-beta1, in a concentration-dependent way, promoted the disruption of follicles, cell spreading, migration and confluency by a mechanism that did not involve cell proliferation. TGF-beta1 strongly activated the production of thrombospondin-1 and (alpha)vbeta3 integrin in a concentration-dependent manner whereas the expression of thyroglobulin was unaffected. Anisomycin, an inhibitor of protein synthesis, inhibited the effect of TGF-beta1 on cell organization. Thrombospondin-1 reproduced the effect of TGF-beta1. In the presence of thrombospondin-1 cells did not organize in follicle-like structures but, in contrast, spreaded and reached confluency independently of cell proliferation. This effect is suppressed by an RGD-containing peptide. The adhesive properties of thrombospondin-1 for thyroid cells were shown to be mediated by both the amino-terminal heparin-binding domain and the RGD domain of thrombospondin-1. Adhesion was shown to involve (alpha)vbeta3 integrin. The results show that TGF-beta1 exerted an influence upon function and behaviour of follicle cells partly mediated by the synthesis of thrombospondin-1 and of its receptor (alpha)vbeta3 integrin.

All-trans-retinoic acid enhances collagen gene expression in irradiated and non-irradiated hairless mouse skin.

All-trans-retinoic acid (t-RA) can repair some of the tissue damage caused by chronic exposure of skin to UV radiation. In the present study, we have investigated its effect on collagen and collagenase gene expression in hairless mouse skin. Hairless mice (SKH-hr 1) were irradiated dorsally with increasing doses of UVB radiation (total, 4.8 J cm-2) for 10 weeks. The animals were then topically treated with 0.05% t-RA dissolved in a vehicle or with the vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatment. In our experimental conditions, UVB irradiation alone induced no changes in type I, III and VI collagen mRNA levels in dorsal and ventral skin. The mRNA level of collagenase I was also unchanged. Topically applied t-RA increased the steady state levels of type I and III collagen mRNA in irradiated and non-irradiated dorsal skin. The mean increase was about 2.2- and 2.7-fold in non-irradiated skin and 2.4- and 2.5-fold in irradiated skin for type I and III collagen mRNA respectively. The increase in irradiated skin was partly due to the vehicle alone, which exerted a stimulating effect on the steady state levels of alpha 1(I) and alpha 1(III) mRNA. The mRNA level of type VI collagen was also significantly increased by t-RA, but only in irradiated skin. The mRNA level of collagenase was significantly decreased only in irradiated t-RA-treated skin. In addition, t-RA exerted a systemic effect because the mRNA levels of collagen were enhanced by factors of 1.9 and 2.5 for alpha 1(I) and 2.0 and 2.0 for alpha 1(III) in the ventral skin of irradiated and non-irradiated animals respectively. This study leads to the conclusion that topical t-RA exerts directly and/or indirect effects on the expression of collagen genes in irradiated and non-irradiated hairless mouse skin.

Isolation of a developmentally-regulated expressed sequence tag from bladder tissue using the mRNA differential display.

In order to gain insight into the molecular and cellular events that govern the structural and the functional properties in developing organs, we have conducted a study to identify genes that have a temporally-restricted expression in the bladder wall during fetal development. We utilized the mRNA differential display technique and compared the pattern of gene expression during the first, the second and the third trimester of gestation. We cloned and sequenced a cDNA fragment (bld-10) which was expressed during the second and third trimester but consistently absent during the first trimester. The bld-10 sequence is not related to any known gene in the GenBank database but has significant homology (89%) with human expressed sequence tag (EST) that has been cloned from human fetal heart and brain libraries. When used in Northern-blot hybridization as a probe, the fragment bld-10 generates two hybridization signals of 3.1 and 4.0 kb, that are minimally expressed during the first trimester of gestation and upregulated in the second and third trimester. Differential expression of this gene may be responsible for some of the profound changes which occur during organ development.

Glutamine increases collagen gene transcription in cultured human fibroblasts.

We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells.

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.

In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.