摘要:

AbstractThe purpose of this study was to examine the effects of IL‐1β on integrin expression in MG‐63 human osteosarcoma cells. Human recombinant IL‐1β (rIL‐1β) produced significant increases in both α2‐ and α5‐subunit mRNA levels, as well as a smaller increase in αv‐subunit mRNA. In contrast, IL‐1β decreased α4‐subunit mRNA levels by approximately 30% relative to untreated controls. These findings suggest that human IL‐1β differentially regulates expression of integrins. When cultures were treated with both IL‐1β and the cyclooxygenase inhibitor, indomethacin, the expression of α2‐, α5‐, and αv‐subunit mRNA levels were dramatically increased relative to untreated controls; co‐treatment with 0.5 mM prostaglandin E2(PGE2) partially reversed this effect. Indomethacin alone did not affect integrin mRNA levels.Treatment with IL‐1β or IL‐1β + indomethacin also induced significant changes in MG‐63 morphology (i.e., increased cell elongation) and increased the ability of cells to contract collagen gels. PGE2reversed the above effects on cell morphology and gel contraction.These findings indicate that (a) IL‐1β differentially regulates the expression of integrins and (b) that PGE2, which is induced by IL‐1β, may provide a negative feedback loop which counteracts the stimulatory effect of IL‐1β on integrin gene expression. It is suggested that products of inflammation may affect cell behavior by di

ISSN:0021-9541    

DOI:10.1002/jcp.1041490202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe Na+,K+‐ATPase α1, α2, and α3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE)(Martin‐Vasallo et al., J. Cell. Physiol.,141: 243‐252, 1989; Ghosh et al., J. Biol. Chem.,265:2935‐2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K+‐ATPase α (α1, α2, α3) subunit genes with the pattern of expression of the Na+,K+‐ATPase β (β1, β2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as (1) the anterior region of the pars plicata, near the iris; (2) the middle region of the pars plicata; and (3) the posterior region of the pars plana, near the ora serrata. Using isoform‐specific cDNA probes and antibodies for the Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of α and β isoforms detected along the NPE cell layer suggests a gradient of α1, α2, α3, β1, and β2 mRNAs and polypeptides that correlates with decreasing Na+,K+‐ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the α (α1, α2, α3) and β (β1, β2) Na,K‐ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with α1, α2, α3, and β1 antibodies, although staining with β2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to α1 and β1, while no staining signals were detected with the rest of the antibodies (i.e. α2, α3, and β2). Our results support the conclusion that the three α and two β isoforms of the Na+,K+‐ATPase are differentially expressed in the two cell layers that make up the CE. These results also suggest that the expression of the Na+,K+‐ATPase α and β subunit isoforms

ISSN:0021-9541    

DOI:10.1002/jcp.1041490203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAdherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence‐dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell‐derived extracellular matrix or a denatured collagen/fibronectin‐coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2(PGE2) and thromboxane B2(TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin‐coated wells demonstrated significantly less cytolytic activity (15 ± 2% lysis) when compared with either tissue culture plastic adherent PM (43 ± 7% lvsis) or PM adherent to extracellular matrix (59 ± 11% lysis). PM adherent to extracellular matrix released twofold more TxB2than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2than cells adherent to tissue culture plastic or 80% more PGE2than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 ± .9 nmoles/106PM) than PM adherent to either tissue culture plastic (43 ± 1 nmoles/106PM) or the extracellular matrix (60 ± 0.5 nmoles/106PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2reduced superoxide production in a dosedependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during

ISSN:0021-9541    

DOI:10.1002/jcp.1041490204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis‐independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5°C, they became thermotolerant to a heat treatment at 45.5°C administered 12 hr later. The thermotolerance ratio at 10−3isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2–2 μg/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 μg/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 μg/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance. development by treatment with a combination of 2 μg/ml tunicamycin and 10 μg/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis‐independent thermotolerance, is not correlated with increased glycosylation of the

ISSN:0021-9541    

DOI:10.1002/jcp.1041490205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2(PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell‐laminin interactions. As has been reported for many other tumor cells, laminin and the laminin‐derived peptide PA22‐2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cellsin vitro.We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22‐2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose‐dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22‐2‐mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2receptor modulates tumor cell adhesion to laminin which may subsequently affect thein vivoprocess

ISSN:0021-9541    

DOI:10.1002/jcp.1041490206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have reported earlier the isolation of two recessive, serum‐ and anchoragedependent revertants (R116 and R260) from a c‐H‐ras oncogene‐transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity.We report here that neither revertant could be re‐transformed by the K‐rasor N‐rasoncogene (though they could be re‐transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double‐minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage

ISSN:0021-9541    

DOI:10.1002/jcp.1041490207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA previously described chondrocyte alkaline phosphatase induction factor (CAP‐IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS‐PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye‐ligand affinity (Affi‐Gel Blue and Reactive Green‐19 agarose) and hybroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine‐labeled cellular proteins after 3 day treatment, this highly purified CAP‐IF increases the level of AP and certain other membrane proteins 2‐ to 3‐fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N‐terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)‐inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP‐IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+did not reactivate the EDTA‐treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8‐fold during 3 day exposure. Because of the very high homology between fetuin and the A‐chain of α2‐HS glycoprotein, we also tested and found that α2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP‐inducing activity resides in a labile, cystatin/Zn2+‐binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal g

ISSN:0021-9541    

DOI:10.1002/jcp.1041490208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF‐AB and PDGF‐BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF‐AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF‐AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF‐AA were absent. PDGF‐AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti‐Type α and β PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF α and β receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of β receptors. These results indicate that cells possessing high levels of PDGF β receptors only are capable of responding equally well to e

ISSN:0021-9541    

DOI:10.1002/jcp.1041490209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractRetinoic acid and dexamethasone have antagonistic effects on epidermal growth factor (EGF) receptor expression in fetal rat lung (FRL) cells: Receptor synthesis is enhanced by retinoic acid and reduced by dexamethasone. In the presence of actinomycin D, neither agent has the capacity to modify receptor synthesis or125I‐EGF binding capacity. Northern blot analysis demonstrates a tenfold increase in EGF mRNA following retinoic acid treatment and a 60% decrease in receptor message levels after dexamethasone treatment. To dissect the mechanisms of these effects, the expression of mRNA was separated from effects requiring protein synthesis by the use of cycloheximide and actinomycin D. Ligand binding, EGF receptor protein synthesis, and mRNA levels were measured in cultures of FRL cells that were incubated with retinoic acid or dexamethasone in the presence of cycloheximide, then washed and reincubated with fresh media containing actinomycin D, but not retinoic acid, dexamethasone, or cycloheximide. The results demonstrate that dexamethasone reduces the expression of EGF receptor mRNA in the absence of protein synthesis. In contrast, the mechanism by which retinoic acid increases the expression of EGF receptor mRNA requires protein synthesis. These data indicate that, in FRL cells, dexamethasone negatively regulates EGF receptor mRNA in a direct manner, while retinoic acid controls transcription of an intermediate protein, possibly a transcription factor, that subsequently increases transcription of receptor messag

ISSN:0021-9541    

DOI:10.1002/jcp.1041490210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAfter a 3 to 6 hour incubation, addition of progesterone (the most effective), insulin‐like growth factor 1 (IGF‐1; the second most effective), or insulin induces meiotic cell division inXenopusoocytes. Measurement of an endogenous activator of protein kinase C, sn‐1,2‐diacylglycerol (DAG), by an enzymatic method recording mass demonstrates that all three hormones alter DAG levels. Five seconds after addition, only progesterone transiently reduces DAG levels by about 25%. At 15 minutes after addition, all three hormones produce a peak of DAG (115% to 160% of control values), with the more effective hormones producing a larger increase in DAG. Insulin produces the smallest DAG increase, but the DAG release is longer lasting. Finally, all three hormones induce a second peak in DAG levels just before white spot appearance (at 0.85 GVBD50, where 1.0 GVBD50is when 50% of the cells have divided). With these data and since an activator of protein kinase C, a phorbol ester, has been found to induce meiosis, the kinase may play a role in early proliferative events at the plasma membrane and in late events at the

ISSN:0021-9541    

DOI:10.1002/jcp.1041490211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY