摘要:

AbstractThe knowledge of transforming growth factor (TGF)‐β receptors has greatly progressed in the recent years. TGF‐β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF‐β to its signaling receptors. In addition, four other proteins that bind TGF‐β1 or TGF‐β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF‐β receptors remain an enigma. TGF‐β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF‐β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF‐β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF‐β1. Using [3H]‐inositol labeling and Triton X‐114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI‐PLC) or by exposure to TGF‐β, supporting that a PI‐anchored molecule gives rise to IPG by TGF‐β‐induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF‐β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effec

ISSN:0021-9541    

DOI:10.1002/jcp.1041550302

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe influence of zinc (Zn) availability on thymidine kinase mRNA concentration has been investigated in cells in which production of the mRNA was regulated by either truncated thymidine kinase promoters or by the SV40 early promoter. Thymidine kinase mRNA concentrations were decreased by low Zn availability even when the promoter was truncated to 80 bp but not when it was replaced by the SV40 promoter. However, thymidine incorporation by the SV40 cells was still sensitive to lack of Zn, suggesting a second Zn‐sensitive process involved in commitment to S phase. The increase in histone H3 mRNA production prior to S phase was not inhibited by lack of Zn leading to a preferential increase in this mRNA in exponentially growing cells deprived of Zn. © 1993 Wiley‐Liss,

ISSN:0021-9541    

DOI:10.1002/jcp.1041550303

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

年代:1993

数据来源: WILEY

摘要:

AbstractIt has been shown that no relation exists between [Ca2+]iand hyperthermic cell killing, although heat‐induced increase of [Ca2+]ican be observed in some cell lines. When ionophores are used, dose‐dependent rises in [Ca2+]imay be found. Beyond a certain threshold of ionophore‐induced increases in [Ca2+]i, cells may be killed. Different threshold levels of [Ca2+]iexist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca2+]iand heat toxicity, this synergism can be explained as heat enhanced Ca2+toxicity. In the current report, it is shown that both ionophore‐induced Ca2+toxicity (37°C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca2+]i. With ionomycin concentrations up to 15 μM, no increase in [Ca2+]iwas seen in cells maintained in medium without Ca2+. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041550304

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

年代:1993

数据来源: WILEY

摘要:

AbstractDegradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen‐coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of3H‐Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead‐labeled phagosomes in single, pre‐mitotic cells demonstrated that>90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis‐inducing drugs nifedipine or dilantin. These cultures exhibited marked (15–75%), dose‐dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis. © 1993 W

ISSN:0021-9541    

DOI:10.1002/jcp.1041550305

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

年代:1993

数据来源: WILEY

摘要:

AbstractThis report documents characterization of five osteogenic cell subpopulations of bone marrow stroma. The clonally derived cell lines were isolated from the parental line MBA‐15 known to express osteoblastic‐associated features in vitro and to form bone in vivo. The latter, presumably “arrested” at a particular stage along the osteogenic lineage, are useful models to study the processes involved in the differentiation of bone forming cells. The clones differ in their morphology, proliferation rate, quantities and distribution of extracellular matrix proteins, levels of alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone and/or prostaglandin E. These properties have been retained during prolonged growth and subculturing through many passages. MBA‐15.4 is a presumptive preosteoblast with a fibroblast‐like appearance; it proliferates rapidly, synthesizes equal amounts of collagen and noncollagenous proteins, and produces constitutively low levels of alkaline phosphatase. This clone has PGE2‐stimulated adenylate cyclase activity and a very low constitutive response to PTH. On the other hand, MBA‐15.6 has a large polygonal morphology with limited proliferative potential, synthesizes twice as much noncollagenous proteins as collagen, has high alkaline phosphatase activity, and responds strongly to PTH. The characteristics of the other clones place them between these two categories. The effects of 10−7M dexamethasone or 10−12–10−8M 1,25 dihydroxyvitamin D3on growth and differentiation further strengthen the variance between these clones. The different in vitro characteristics of the various clones were directly reflected in their bone formation ability in vivo. When transplanted under the renal capsule, MBA‐15.33 formed a thick fibrous tissue, MBA‐15.4 formed small foci of bone, and MBA‐15.6 formed massive woven bone at the same period of

ISSN:0021-9541    

DOI:10.1002/jcp.1041550306

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

年代:1993

数据来源: WILEY

摘要:

AbstractPrimary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor‐β (TGFβ) and to be growth inhibited by exogenous TGFβ. The purpose of the present studies was to determine whether the endogenous TGFβ(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGFβ1 and TGFβ2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8‐and 3‐fold increases in colony formation and cell number, respectively, above control IgG‐treated cultures. Antibody dose‐response experiments revealed that TGFβ2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGFβs were acting locally. Anti‐TGFβ1 treatment of cultures at various stages of growth resulted in 1.2–1.7‐fold increases in DNA synthesis above controls, whereas anti‐TGFβ2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7‐ and 2.5‐fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGFβs inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4–70‐fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGFβ1 and TGFβ2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of en

ISSN:0021-9541    

DOI:10.1002/jcp.1041550307

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

年代:1993

数据来源: WILEY

摘要:

AbstractHydrogen peroxide (H2O2) overload may contribute to cardiac ischemia‐reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H2O2‐induced oxidative stress on heart‐muscle purine and pyrimidine nucleotides and high‐energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H2O2elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de‐energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 μM to 1.0 mM H2O2. At the highest H2O2concentration, 5 min was sufficient to elicit appreciable cardiomyocyte highenergy phosphate loss, the extent of which could be limited by prompt elimination of H2O2from the culture medium. Only H2O2dismutation completely prevented ATP loss during H2O2‐induced oxidative stress, whereas various freeradical scavengers and metal chelators afforded no significant ATP preservation. Exogenously‐supplied catabolic substrates and glycolytic or tricarboxylic acidcycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de‐energization during H2O2‐induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H2O2‐induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de‐energization in response to H2O2overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post‐ischemic heart

ISSN:0021-9541    

DOI:10.1002/jcp.1041550308

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

年代:1993

数据来源: WILEY

摘要:

AbstractActivation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD‐phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of32P into cellular proteins using SDS‐PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+]<10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus‐induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F‐actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041550309

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

年代:1993

数据来源: WILEY

摘要:

AbstractTwo complementary experimental methods have been used to examine mitogen‐induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen‐induced changes in [Ca++]iand transmembrane potential. Activation of human B cells with anti‐μ antibodies resulted in a biphasic rise in [Ca++]i, the second phase being mediated by the influx of extracellular Ca++. Ca++influx was inhibited by high [K+]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti‐μ antibodies to the bath solution, inward currents were observed at negative voltages. Whole‐cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than −40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3into the intracellular solution, suggesting that IP3generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti‐μ and IP3induced currents were blocked by 1 mM La+++, which is known to block Ca++channels. These results strongly support the presence of membrane Ca++channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++influx. These mechanisms work in concert to regulate the level of [Ca++]iduring the early phases of human B cell activation. © 199

ISSN:0021-9541    

DOI:10.1002/jcp.1041550310

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have examined nucleoside transport (NT) in a cell line derived from primary day 7 murine bone marrow macrophages (S1 macrophages) in response to the macrophage growth factor, colony‐stimulating factor 1 (CSF‐1). Adenosine and uridine transport in quiescent S1 macrophages occurred primarily by two facilitated diffusional routes, one that was sensitive and one that was relatively resistant to the inhibitor nitrobenzylthioinosine (NBMPR). Addition of CSF‐1 to quiescent cultures resulted in increased adenosine and uridine transport with biphasic kinetics with respect to the cell cycle. Basal NT activity was elevated (about twofold) within 15 min of CSF‐1 addition, returned to near basal levels by 1 h, and then increased again (three‐ to fourfold) 8–12 h later, returning again to basal levels by 48 h post CSF‐1 stimulation. We propose that the large increase in NT activity at 8–12 h corresponded with the time when cultures synchronously began to enter the S phase of the cell cycle. In addition to these changes in the absolute rates, the proportions of NBMPR‐sensitive and NBMPR‐insensitive transport also change after CSF‐1 addition. Quiescent cultures exhibited primarily NBMPR‐insensitve transport while logrithmically growing cultures exhibited primarily NBMPR‐sensitive nucleoside transport activity. The increase in the NBMPR‐sensitive component of the transport process paralleled a similar increase in the number of high‐affinity NBMPR binding sites, suggesting that the mechanism for upregulating NBMPR‐sensitive NT activity involves increases in the number of NBMPR‐sensitive transporter sites. Interestingly, we were unable to detect Na+‐dependent concentrative uptake of adenosine, uridine, or formycin‐B either in the S1 macrophage cell line or in primary (day 7) murine macrophages. Thus these bone marrow derived macrophages did not display the characteristically large Na+‐dependent transport systems observed by others in peritoneal macrophages, implying that these two populations of macrophages are, indeed, functi

ISSN:0021-9541    

DOI:10.1002/jcp.1041550311

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

年代:1993

数据来源: WILEY