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1.
The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen I, and Matrigel). A 24 h exposure of the cells to 10-6 M or 10-7 M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen I, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10-6 M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGFβ, and IGF-I). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured. © 1996 Wiley-Liss, Inc. 相似文献
2.
D. Benayahu A. Fried A. Shamay N. Cunningham S. Blumberg S. Wientroub 《Journal of cellular biochemistry》1994,56(1):62-73
The effects of retinoic acid (RA) on the expression of osteoblastic-related cell makers was examined. A marrow and osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constituentively express mRNA encoding for procolllagen a2 (1), osteonectin, osteopontin, biglycan and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity dispalyed a different pattern. MBA-15.4, a presosteoblast cell ine, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor β (TGFβ). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic increase in MBA-15.6 cell responses to PTH and PGE2 but no significant effects could be observed in other clonal lines. 相似文献
3.
K. Traianedes K. W. Ng T. J. Martin D. M. Findlay 《Journal of cellular physiology》1993,157(2):243-252
The aim of this study was to determine the role of ECM components of bone in regulating the differentiation and function of cells of the osteoblast lineage. Rat UMR 201 cells, phenotypically preosteoblast, were plated onto plastic tissue culture dishes or dishes coated with gelled type I collagen or reconstituted basement membrane (matrigel). Acute cell attachment assays showed that cells adhered to substrates in the following order: collagen > matrigel ? plastic. Proliferation rate up to 96 hr were similar on each substrate. However, if cells were treated with 10?6 M retinoic acid (RA), proliferation rates were reduced compared with control for cells grown on collagen and matrigel but not on plastic. Morphological changes were matrix-specific; in subconfluent cultures, long thin processes were seen with cells grown on collagen and a pattern of interconnecting cell processes formed when cells were plated on matrigel. Striking differences were observed in the constitutive or RA-induced gene expression of cells grown on the different substrates. When cells plated on collagen were treated with RA, induction of mRNA for alkaline phosphatase (ALP) as well as ALP enzyme activity were much less than with cells grown on plastic. In contrast, RA treatment induced osteopontin (OP) mRNA expression more strongly in cells plated on collagen compared with plastic within 24 hr and this was maintained for 72 hr. RA treatment produced a two fold increase of pro-α 1(I) collagen mRNA in cells grown on plastic and matrigel but not in cells grown on collagen. Growth on collagen produced changes in the way UMR 201 cells responded to RA from which they did not fully recover in subsequent 48-hr growth periods on plastic. These results indicate that ECM components regulate the function of and are capable of modulating RA-induced differentiation of preosteoblasts. © 1993 Wiley-Liss, Inc. 相似文献
4.
A. K. Brar G. R. Frank R. G. Richards A. J. Meyer C. A. Kessler M. I. Cedars D. J. Klein S. Handwerger 《Journal of cellular physiology》1995,163(1):30-37
The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE2 stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc. 相似文献
5.
D Zipori D Duksin M Tamir A Argaman J Toledo Z Malik 《Journal of cellular physiology》1985,122(1):81-90
A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo. 相似文献
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8.
Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis 总被引:20,自引:0,他引:20
Hironaka K Sakaida I Matsumura Y Kaino S Miyamoto K Okita K 《Biochemical and biophysical research communications》2000,267(1):290-295
Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis. 相似文献
9.
M Asada-Kubota 《Journal of structural biology》1990,103(2):113-123
The expression of intracellular and surface polarity in animal pole cells of Xenopus embryos (stage 10) cultured on various substrata was studied by electron microscopy. When animal pole cells of Xenopus embryos were cultured on type I collagen- or gelatin-coated dishes until control embryos reached stage 23, the cells in confluent layers expressed an apical-basal polarity so that the apical surface membrane domain faced the culture medium. However, the cells in confluent layers cultured on naked plastic dishes were suppressed to express intracellular and surface polarity. In addition, single attached cells which were formed by being sparsely plated neither spread on any substrata nor displayed the apical-basal polarity perpendicular to the dishes. These results indicate that the expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos requires not only cell-cell contact but also an adhesive substrata such as type I collagen or gelatin. 相似文献
10.
Extracellular matrix-dependent regulation of Fas ligand expression in human endometrial stromal cells. 总被引:9,自引:0,他引:9
Belgin Selam Umit A Kayisli Juan A Garcia-Velasco Aydin Arici 《Biology of reproduction》2002,66(1):1-5
Interaction between endometrial stromal cells and extracellular matrix (ECM) components has a crucial role in the development of endometriosis. Endometrial stromal cells attach to the mesothelial surface of peritoneum by means of integrins during their initial implantation and growth in endometriosis. Similarly, interaction between integrin and the extracellular matrix is also crucial for the remodeling of the endometrium during early pregnancy. We hypothesized that adhesion of endometrial stromal cells to the extracellular matrix could suppress the immunologic reaction to implanting endometrial cells by inducing the expression of Fas ligand (FasL), a mediator of the apoptotic pathway. Western blot analysis of human endometrial stromal cells plated onto fibronectin, laminin, and collagen IV revealed higher levels of FasL protein expression compared with endometrial stromal cells that plated to BSA-coated plates (control). Immunocytochemistry results from endometrial stromal cells plated to extracellular matrix proteins demonstrated a similar up-regulation of FasL expression. Eutopic endometrial stromal cells from women with endometriosis demonstrated higher FasL expression on control plates and those coated with extracellular matrix proteins compared with those from women without endometriosis. Disruption of actin cytoskeleton in endometrial stromal cells by treatment with cytochalasin D blocked the increase of FasL protein expression that occurred in response to adhesion to the extracellular matrix. These results suggest that attachment of endometrial stromal cells during retrograde menstruation to a new environment such as peritoneum with increased expression of laminin, fibronectin, and collagen IV could lead to an increase in FasL expression. Induction of FasL expression by adhesion of endometrial stromal cells to the extracellular matrix may take part in the development of a relative immunotolerance by inducing apoptosis of cytotoxic T lymphocytes, which will allow further development of ectopic implants. 相似文献
11.
Murakami Y Hirata H Miyamoto Y Nagahashi A Sawa Y Jakt M Asahara T Kawamata S 《Mechanisms of development》2007,124(11-12):830-839
It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system. 相似文献
12.
SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway 总被引:15,自引:2,他引:13
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《The Journal of cell biology》1993,121(6):1433-1444
SPARC (osteonectin/BM40) is a secreted protein that modifies the interaction of cells with extracellular matrix (ECM). When we added SPARC to cultured rabbit synovial fibroblasts and analyzed the secreted proteins, we observed an increase in the expression of three metalloproteinases--collagenase, stromelysin, and the 92-kD gelatinase-- that together can degrade both interstitial and basement membrane matrices. We further characterized the regulation of one of these metalloproteinases, collagenase, and showed that both collagenase mRNA and protein are upregulated in fibroblasts treated with SPARC. Experiments with synthetic SPARC peptides indicated that a region in the neutral alpha-helical domain III of the SPARC molecule, which previously had no described function, was involved in the regulation of collagenase expression by SPARC. A sequence in the carboxyl-terminal Ca(2+)-binding domain IV exhibited similar activity, but to a lesser extent. SPARC induced collagenase expression in cells plated on collagen types I, II, III, and V, and vitronectin, but not on collagen type IV. SPARC also increased collagenase expression in fibroblasts plated on ECM produced by smooth muscle cells, but not in fibroblasts plated on a basement membrane-like ECM from Engelbreth-Holm-Swarm sarcoma. Collagenase was induced within 4 h in cells treated with phorbol diesters or plated on fibronectin fragments, but was induced after 8 h in cells treated with SPARC. A number of proteins were transiently secreted by SPARC-treated cells within 6 h of treatment. Conditioned medium that was harvested from cultures 7 h after the addition of SPARC, and depleted of residual SPARC, induced collagenase expression in untreated fibroblasts; thus, part of the regulation of collagenase expression by SPARC appears to be indirect and proceeds through a secreted intermediate. Because the interactions of cells with ECM play an important role in regulation of cell behavior and tissue morphogenesis, these results suggest that molecules like SPARC are important in modulating tissue remodeling and cell-ECM interactions. 相似文献
13.
Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion 总被引:13,自引:3,他引:10
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J F Cajot W D Schleuning R L Medcalf J Bamat J Testuz L Liebermann B Sordat 《The Journal of cell biology》1989,109(2):915-925
A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype. 相似文献
14.
Effect of extracellular matrix on prolactin secretion and mRNA accumulation in GH3 cells 总被引:1,自引:0,他引:1
The matrix upon which cells grow affects their morphology, growth rate, response to external stimuli, and protein synthesis. GH3 cells, a well-characterized rat pituitary tumor cell line, synthesize and secrete growth hormone and prolactin (Prl). These cells are rounded, attach loosely, and form clumps when plated on plastic. GH3 cells plated on an extracellular matrix (ECM) from bovine corneal endothelial cells become flattened and strongly adherent to the culture dish, and have an initial increased rate of proliferation. Cells cultured on plastic have a 48-hr lag period before the start of cell division; this can be shortened by increasing the concentration of serum in the medium. Since GH3 cells store little Prl, hormone release is a good index of Prl synthesis. Prl secretion from cells cultured on extracellular matrix is twice as great as from cells cultured on plastic. The increase in Prl secretion from cells grown on extracellular matrix paralleled by a concomitant increase in the accumulation of prolactin mRNA. Cells cultured on plastic secrete more Prl in response to TRH stimulation than do cells cultured on ECM. Cells grown on either surface were unresponsive to dopamine. Thus, culturing cells on ECM may change their morphology and affect the synthesis and regulation of specific cellular proteins and their mRNAs. 相似文献
15.
Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix 总被引:26,自引:0,他引:26
E G Schuetz D Li C J Omiecinski U Muller-Eberhard H K Kleinman B Elswick P S Guzelian 《Journal of cellular physiology》1988,134(3):309-323
Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions. 相似文献
16.
Takashi Hoshiba Hongxu Lu Naoki Kawazoe Tomoe Yamada Guoping Chen 《Biotechnology progress》2013,29(5):1331-1336
Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013 相似文献
17.
Zhe Liu Xuanjia Dong Zhijie Cao Shaowei Qiu Yihui Li Mengjun Zhong Zhenya Xue Yingxi Xu Haiyan Xing Kejing Tang Zheng Tian Min Wang Qing Rao Jianxiang Wang 《Experimental biology and medicine (Maywood, N.J.)》2021,246(15):1750
Alternative splicing (AS) is a critical regulatory process of gene expression. In bone marrow microenvironment, AS plays a critical role in mesenchymal stem cells fate determination by forming distinct isoforms of important regulators. As a spliceosome factor, U2AF1 is essential for the catalysis of pre-mRNA splicing, and its mutation can cause differential AS events. In the present study, by forced expression of mutant U2AF1 (U2AF1S34F) in the mouse bone marrow stroma OP9 cells, we determine AS changes in U2AF1S34F transduced OP9 cells and investigate their role in stroma cell biological functions. We find that abundant differential RNA splicing events are induced by U2AF1S34F in OP9 cells. U2AF1S34F causes increased generation of hydrogen peroxide, promotes production of cytokines and chemokines. U2AF1S34F transduced OP9 cells also exhibit dysfunction of mitochondria. RNA-seq data, gene ontology (GO), and gene set enrichment analysis reveal that differentially expressed genes downregulated in response to U2AF1S34F are enriched in peroxisome component and function. U2AF1S34F can also cause release of hydrogen peroxide from OP9 cells. Furthermore, we investigate the influence of U2AF1S34F-induced oxidative stress in stromal cells on hematopoietic cells. When co-culturing mouse bone marrow mononuclear cells with OP9 cells, the U2AF1S34F expressing OP9 cells induce phosphorylation of histone H2AX in hematopoietic cells. Collectively, our results reveal that mutant U2AF1-induced differential AS events cause oxidative stress in bone marrow stromal cells and can further lead to DNA damage and genomic instability in hematopoietic cells. 相似文献
18.
A M Choi D R Olsen K G Cook S F Deamond J Uitto S A Bruce 《Journal of cellular physiology》1992,151(1):147-155
Increasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation-like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation-associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady-state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen alpha 1III, and procollagen alpha 1I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, alpha 1III, and alpha 1I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of alpha 1III mRNA as the cells reached the end of their in vitro proliferative life span, and then alpha 1I when the cells entered the postmitotic senescent phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up-regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair. 相似文献
19.
The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function. 相似文献
20.
Liang Qiao Geoffrey C. Farrell 《In vitro cellular & developmental biology. Animal》1999,35(7):417-424
Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high
and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were
greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei
in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures
but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on
collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis.
The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells
were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen
1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures,
aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures
of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated,
at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent
manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell
density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes. 相似文献