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1.
The expression of fibronectin (FN) mRNA isoforms generated by alternative splicing of the EDA region was studied by dot-blot and in situ hybridization, using specific FN cDNA probes, in skin fibroblasts from controls and Ehlers-Danlos Syndrome (EDS) patients (types III, IV, VII and non classified). An Image Analysis program was used for the quantitative evaluation and comparison of FN mRNAs levels in the different cell strains. The in situ hybridization analysis showed that FN mRNAs are homogeneously expressed in all cells of each fibroblast strain analyzed. While in control fibroblasts about 70% of FN mRNA isoforms contain the EDA region (EDA+ FN mRNAs), in EDS fibroblasts this fraction is reduced up to about 30%. This indicates that in the EDS fibroblasts analyzed a deregulation of the alternative splicing processes acting at the EDA region takes place.  相似文献   

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An Image Analysis program was used for the quantitative evaluation and comparison of the fibronectin (FN) mRNA detected by dot-blot and in situ hybridization in different cell lines. These techniques were applied for the evaluation of FN mRNA synthesized by human normal fibroblasts (Flow 7000) and by four tumour-derived cell lines (HeLa, epithelioid carcinoma; 8387, fibrosarcoma; RD, rhabdomyosarcoma; SK Hep-1, hepatocarcinoma). Dot-blot analysis showed that the cell types analysed synthesize different levels of FN mRNA. Flow 7000 are the highest producers while HeLa the lowest. In situ hybridization confirmed these results and furthermore showed that while Flow 7000, 8387 and HeLa cells synthesized homogeneous levels of FN mRNA, RD and SK Hep-1 could be subdivided into two populations expressing high or low levels of FN mRNA. The combined analysis of dot-blot, in situ hybridization and Image Analysis allowed the quantitation of the number of FN mRNA molecules expressed by single cells. This approach is therefore an invaluable tool when evaluating mRNA expression in heterogeneous cell populations like tumour-derived cell lines, during cell cycle or in histological tissue sections.  相似文献   

3.
The effect of dexamethasone (DEX) on the expression of fibronectin (FN), proalpha(1)(I) collagen (Col1), integrin alpha(2), alpha(5)and beta(1)subunits mRNAs, were studied by quantitative in situ hybridization (ISH) with radiolabelled probes in relationship with the organization of the extracellular matrix (ECM) of FN in human skin fibroblasts. In particular, two fibroblast strains were analysed, one derived from a control donor, typically organizing a rich ECM of FN, and the other from a patient affected by Ehlers-Danlos syndrome (EDS), which did not assemble the FN-ECM. Treatment of both fibroblast strains with 10(-7) m DEX slightly enhanced the level of FN mRNA (by about 1.5-fold), did not influence the level of alpha(5)subunit mRNA and reduced Col1, alpha(2)and beta(1)integrin subunits mRNAs by 2-3-fold. These results show that, in these cells, DEX coordinately downregulates the expression of Col1 and its specific integrin alpha(2)beta(1). Moreover, DEX regulates in a different manner the alpha(5)and beta(1)subunits forming the main FN receptor (FNR) in skin fibroblasts. Immunofluorescence microscopy evidencing the FN-ECM and integrins containing alpha(5)and beta(1)subunits showed that in control cells DEX induced a slight enhancement of the FN-ECM and of the alpha(5)beta(1)receptors patches. Therefore, in these cells the decrease of beta(1)FN receptor subunit mRNA, as well as the decrease of Col1 and its receptor mRNAs, did not influence the FN-ECM assembly. In EDS fibroblasts, DEX decreased the cytoplasmic accumulation of FN and induced the assembly of a rich FN-ECM through the formation of large FNR integrin patches, codistributing with the FN-ECM. We suggest that in EDS skin fibroblasts DEX corrects the defective FN-ECM favouring the sorting and the organization of FN and its alpha(5)beta(1)integrin receptor.  相似文献   

4.
Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.  相似文献   

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Activation of type I collagen genes in cultured scleroderma fibroblasts   总被引:2,自引:0,他引:2  
Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.  相似文献   

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The events occurring during emergence of cells from quiescence ("G0") are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.  相似文献   

10.
Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.  相似文献   

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Regulation of fibronectin (FN) by Platelet-derived Growth Factor-BB (PDGF-BB) and Insulin-like Growth Factor-I (IGF-I) in cultured rat thoracic aortic adventitial fibroblasts were examined. PDGF-BB enhances FN mRNA levels in a time and concentration-dependent fashion. The effect of IGF-I on PDGF-BB-induced FN levels was also examined. There was a larger increase in FN mRNA levels (4-fold) in the cells treated with both IGF-I and PDGF-BB than with either IGF-I (2-fold) or PDGF-BB (2-fold) alone. The effects of PDGF-BB and IGF-I on FN levels were examined. There was a larger increase in FN levels (135%) in the cells treated with both PDGF-BB and IGF-I than with either PDGF-BB (43%) or IGF-I (45%) alone. Regulation of adventitial fibroblast FN may lead to blood vessel diseases and/or angiogenesis.  相似文献   

14.
The nucleotide sequence of five independent cDNA clones, which cover 4843 nucleotides from the poly(A) addition site of human fibronectin (FN) mRNA was determined. The deduced amino acid sequence (1383 residues) covers the COOH-terminal 60% of human FN, spanning the C-terminus, fibrin-, heparin- and cell-binding domains, and shows the exact location of the only two free sulphydryl groups present in each subunit chain. We have recently reported two different FN mRNA species; one of them containing an additional 270 nucleotide insert (ED) that encodes exactly one of the homology type III repeats of the protein. The two mRNAs arise by alternative splicing of a common precursor. S1 nuclease mapping of cDNA/RNA hybrids shows that the expression of the two mRNAs is cell specific. Liver only produces the mRNA without the ED, whereas hepatoma cells, breast tumor cells and normal fibroblasts produce both forms of mRNA. Another area of alternative splicing generating three different FN mRNAs in rat liver has been reported by Schwarzbauer et al (16). We here provide evidence for the existence in human cells of a fourth mRNA species different from the three described in rat liver.  相似文献   

15.
Glucocorticoids decrease the synthesis of type I procollagen mRNAs   总被引:2,自引:0,他引:2  
Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.  相似文献   

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EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.  相似文献   

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The effects of interferon-alpha and interferon-gamma on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-gamma treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-gamma reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-alpha on procollagen mRNA levels was somewhat weaker than that of interferon-gamma. The results suggest potential use of interferon-gamma in treatment and prevention of human fibrotic conditions.  相似文献   

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