bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of protein kinase cα in the induction of carcinoembryonic antigen by transforming growth factor β
Authors: Chakrabarty Subhas , Huang Shuang
Published: February 2005

Previous studies showed that transforming growth factor b̃ (TGFb̃1) regulates the expression of carcinoembryonic antigen (CEA) and CEA‐cross‐reactive glycoproteins (CEA‐GLYs) in human colon carcinoma cells through a signal‐transducing pathway associated with protein kinase C (PKC) (Chakrabarty, J. Cell. Physiol., 1992, 152 494–499). In this study we determined the role of the PKCα isoform in the regulation of CEA and CEA‐GLYs expression by TGFb̃1. Expression of PKCα antisense RNA, through transfection experiments with an antisense PKCα expression vector, resulted in down‐modulation of PKCα RNA and protein expression. TGFb̃1 was unable to stimulate the expression and secretion of CEA in cells in which the expression of PKCα protein was substantially reduced. The ability of TGFb̃1 to stimulate the expression of the 95‐ and 55‐KDa CEA‐GLYs, however, was not affected. We therefore conclude that TGFb̃1 regulates the secretion and expression of CEA through a signal‐transducing pathway associated with PKCα. TGFb̃1 may also regulate the expression of CEA‐GLYs through signal‐transducing pathways associated with other PKC isoforms. © 1995 Wiley‐Liss, Inc.
Esterification of 12(s)‐hydroxy‐5,8,10,14‐eicosatetraenoic acid into the phospholipids of human peripheral blood mononuclear cells: Inhibition of the proliferative response
Authors: Joulain Catherine , Meskini Nadia , Anker Genevieve , Lagarde Michel , Prigent Annie‐France
Published: February 2005

12‐hydroxy‐eicosatetraenoic acid (12‐HETE), the lipoxygenase metabolite of arachidonic acid produced by activated platelets, has been shown to accumulate in peripheral blood mononuclear cells (PBMC) of elderly people. 12‐HETE being antimitogenic for lymphocytes, its accumulation in blood cells might be involved in the well‐known decline in immune function which accompanies aging. Because HETEs have been shown to be rapidly metabolized and/or incorporated into cellular lipids in a variety of cell types, we have investigated the uptake, metabolism, and intracellular distribution of exogenous 12‐HETE by human PBMC. [3H]‐12‐HETE was dose and time dependently incorporated by PBMC and also metabolized to more polar products. These polar metabolites were mainly released extracellularly and only marginally esterfied in phospholipids. Although [3H]‐12‐HETE radiolabel was preferentially associated with phosphatidylcholine, especially, a substantial amount of radiolabel was also found associated with phosphatidylinositol (20‐50% of the labeled phospholipids). The stability of 12‐HETE in the phospholipid pool was comparable to that reported for most other cell types, with 50% of the initial radiolabel being still present after 18 hr. Upon exposure to mitogenic activation, 12‐HETE‐labeled PBMC released unmodified 12‐HETE from phosphatidylinositol. In addition, 12‐HETE dose dependently inhibited the proliferative response of PBMC to Con A stimulation. These results suggest that 12‐HETE esterification in phospholipids might lead to the generation of unusual lipid second messengers with impaired capacity to transduce activation signals, thus decreasing lymphocyte function. © 1995 Wiley‐Liss, Inc.
Phenotypically and karyotypically distinct Madin‐Darby canine kidney cell clones respond differently to alkaline stress
Authors: Wünsch S. , Gekle M. , Kersting U. , Schuricht B. , Oberleithner H.
Published: February 2005

We isolated two cell clones from the wild‐type Madin‐Darby canine kidney cell line (MDCK) that resembles renal collecting duct epithelium. Morphology and karyotypes of the two cell clones were evaluated. The MDCK‐C7 cell clone morphologically resembles principal cells (polygonal cell shape, flat), while the MDCK‐C11 clone resembles intercalated cells (cuboidal cell shape, high). The diploid chromosome number of MDCK‐C7 cells is 83.1 ± 0.2 (n = 139); that for MDCK‐C11 cells is 78.8 ± 0.1 (n = 128). Culture of MDCK‐C7 cells in alkaline medium (pH 7.7) induced irreversible phenotypical and genotypical alterations. transformed MDCK‐C7F cells are characterized by two abnormal (biarmed) chromosomes. In contrast, MDCK‐C11 cells are not phenotypically altered by alkaline stress. In order to elucidate the role of intracellular pH (pHi) in the transformation process, we measured pHi under control conditions (pH 7.4). after 5 min exposure to alkaline stress (“acute experiment,” pH 7.7) and after incubation of the cells in alkaline medium for two weeks (“chronic experiment,” pH 7.7). Under control conditions, MDCK‐C7 cells maintained pHi at 7.14 ± 0.01 (n = 154) and MDCK‐C11 cells at 7.01 ± 0.01 (n = 147). Acute alkaline stress increased pHi of both cell types to similar steady‐state values. Under chronic alkaline stress, MDCK‐C7 cells were unable to maintain intracellular pH within normal limits exhibiting sustained alkalinization, whereas MDCK‐C11 cells could successfully regulate pHi. We conclude that wild‐type MDCK cells consist of two genetically distinct subpopulations with different morphology and function. Only the MDCK‐C7 clone that resembles the principle cell type of renal collecting duct can be transformed by alkaline stress while the MDCK‐C11 clone resists this treatment, due to efficient pHi control mechanisms. © 1995 Wiley‐Liss, Inc.
Proteoglycan biosynthesis is required in BC3H1 myogenic cells for modulation of vascular smooth muscle α‐actin gene expression in response to microenvironmental signals
Authors: Lee Sang Hoon , Yan Hua , Reeser Jonathan C. , Dillman John M. , Strauch Arthur R.
Published: February 2005

Induction of vascular smooth muscle (VSM) α‐actin mRNA expression during cytodifferentiation of mouse BC3H1 myogenic cells coincides with the accumulation of cell surface‐ and extracellular matrix‐associated sulfated proteoglycans. Inhibition of proteoglycan biosynthesis in myogenic cells using an artificial b̃‐D‐xyloside glycosaminoglycan acceptor was accompained by a reduction in cell surface/extracellular matrix proteoglycans and VSM α‐actin mRNA expression while enhanciang the secretion of free chondroitin sulfate/dermatan sulfate and heparan sulfate glycosaminoglycans into the culture medium. Maximum inhibition of VSM α‐actin mRNA expression required that proteoglycan biosynthesis be blocked during the early phase of cytodifferentiation when myoblasts were fully confluent and quiescent. The inhibitory effect of b̃‐D‐xyloside on α‐actin mRNA expression resulted from attenuation at both the transcriptional and post‐transcriptional control points. Sustained proteoglycan biosynthesis was required for induction of VSM α‐actin mRNA in quiescent myoblasts in response to cytodifferentiation‐permissive, substrate‐associated macromolecules (SAM) or upon exposure to soluble serum factors capable of transiently stimulating VSM α‐actin gene transcription. The results suggested that efficient myoblast cytodifferentiation and modulation of VSM α‐actin mRNA levels depended on intact cell surface proteoglycans to convey signals generated as a consequence of cellular interaction with substrate components and serum factors. © 1995 Wiley‐Liss, Inc.
Swelling activates chloride current and increases internal calcium in nonpigmented epithelial cells from the rabbit ciliary body
Authors: Botchkin Lev M. , Matthews Gary
Published: February 2005

Membrane current and [Ca]i in rabbit nonpigmented ciliary body epithelial cells (NPE cells) were monitored with combined patch‐clamp and fura‐2 measurements during cell swelling induced by anisosmotic conditions. In the presence of K‐channel blockers, cell swelling produced an increase in membrane current, accompanied by an increase in [Ca]i. Structural changes in the cell, associated with membrane deformation, may be the cause of the increase in [Ca]i during swelling. The conductance activated by swelling was permeable to CI: it was dependent on the CI concentration gradient across the cell membrane, and it was blocked by the CI‐channel blockers DIDS, SITS, NPPB, and DIOA. Although swelling increased both CI current and [Ca]i, there was no evidence that Ca was involved in the regulation of the CI conductance. Cell swelling activated the current even when [Ca]i was strongly buffered at an elevated level (500 nM) or at a low level (∼0) with internal Ca‐BAPTA/Cs‐BAPTA mixtures. In addition, CI conductance was unaffected when [Ca]i was increased with a Ca ionophore. There was also no evidence that cAMP participates in the regulation of the CI conductance: swelling activation of the current occurred in the presence of cAMP inhibitor (Rp‐cAMP‐S) and cAMP mimic (Sp‐cAMP‐S). The data suggest independent involvement of CI conductance and internal Ca in the regulation of cell volume in NPE cells. © 1995 Wiley‐Liss, Inc.
Modulation of the adhesion of hemopoietic progenitor cells to the rgd site of fibronectin by interleukin 3
Authors: Hardy Cheryl L. , Minguell José J.
Published: February 2005

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell‐extracellular matrix (ECM) interactions and also of cell‐cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin‐mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP‐1 were used. Both of these cell lines have been shown to bind to FN‐coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by α5b̃1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of α5 and one antibody against b̃1 were used to study expression of the integrin. By flow cytometry the expression of α5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP‐1 cells the decrement was from 1.0 to 0.8. The loss of RGD‐mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine‐treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation. © 1995 Wiley‐Liss, Inc.
Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion, steroid metabolism, and responsiveness to growth factors
Authors: Lerner Alejandro A. Colman , Salamone Daniel F. , Chiappe M. Eugenia , Barañao J. Lino
Published: February 2005

A bovine granulosa cell line (BGC‐1) has been obtained by spontaneous immortalization of primary cultures. BGC‐1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compaed BGC‐1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC‐1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC‐1 cultures were undetectable. When BGC‐1 cells and primary cultures were incubated with [3H]‐pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [3H]‐progesterone to 5α‐reduced metabolites was markedly increased in BGC‐1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor‐1 (IGF‐I), and transforming growth factor‐b̃ (TGF‐b̃) on DNA synthesis under serum‐free conditions. Both primary cultures and BGC‐1 cells exhibited a stimulatory response to EGF and IGF‐I on [3H]‐thymidine incorporation. Neither BGC‐1 cells nor primary cultures showed a significant response to TGF‐b̃ when added alone. However, in the presence of a combination of EGF and IGF‐I, TGF‐b̃ displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC‐1 cells even further. The addition of conditioned medium from BGC‐1 cells (BGC‐1‐CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC‐1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues. © 1995 Wiley‐Liss, Inc.
Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells
Authors: Yang Wenbo , Block Edward R.
Published: February 2005

Endothelial cells are critical targets in both hypoxia‐and reoxygenation‐mediated lung injury. Reactive O2 species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H2O2 generation and extracellular H2O2 and O/2 release were measured after exposure to normoxia (room air‐5% CO2), hypoxia (0% O2 ‐95% N‐5% CO2), or hypoxia followed by normoxia or hyperoxia (95% O2‐5% CO2). Exposure to hypoxia results in significant reductions in intracellular H2 O2 formation and extracellular release of H2 O2 and O2 by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley‐Liss, Inc.
Control of matrix synthesis in isolated bovine chondrocytes by extracellular and intracellular pH
Authors: Wilkins Robert J. , Hall Andrew C.
Published: February 2005

The effects of extracellular and intracellular pH on matrix synthesis by isolated bovine chondrocytes were studied using radioisotope incorporation (35SO4 and 3H proline) and fluorescence techniques. Matrix synthesis exhibited a bimodal relation with decreased extracellular pH; with slight reductions (7.4>pH>7.1), synthesis increased (by up to 50%), whereas in more acidic media (pH<7.1), synthesis was inhibited by up to 75% of control levels. The pHi was largely unchanged with extracellular acidity over the range producing stimulation of matrix synthesis but fell when exposed to the more acidic media shown to have an inhibitory action on matrix synthesis. The inhibition of matrix synthesis by lactic acid addition was unaffected by the lactic acid transporter α‐CHC, suggesting H+ transport by this pathway is small. Direct imposition of a sustained intracellular acidosis (pHi = 6.65) using ammonium prepulse with amiloride inhibited matrix synthesis by about 20%. These results show that matrix synthesis by chondrocytes was affected by extracellular pH, an action which could not be entirely explained by changes to pHi. © 1995 Wiley‐Liss, Inc.
Release from quiescence stimulates the expression of integrin α5β1 which regulates DNA synthesis in human fibrosarcoma HT1080 cells
Authors: Wang Danhui , Birkenmeier Thomas M. , Yang Junhua , Venkateswarlu Srinivas , Humphrey Lisa , Brattain Michael G. , Sun Luzhe
Published: February 2005

We show that integrin α5 subunit expression is stimulated when human fibrosarcoma HT1080 cells are released from quiescence. The α5 subunit mRNA level in quiescent HT1080 cells was increased 24 hr after their release by 10% fetal bovine serum‐containing medium reaching a maximum of 2.5 fold on day 2. Similar levels of induction of cell‐surface α5 subunit protein as well as b̃1 subunit protein were also observed. This resulted in a significant increase of cell attachment to fibronectin. The serum stimulation also increased α5 subunit promoter activity by twofold which was protein synthesis independent. Subsequent deletion of α5 subunit promoter DNA showed that the cis‐element responsible for the activation is located between ‐ 92 bp and the transcription start site. The promoter activity was not induced until 12 hr after the release. Comparison of the effect of a serum‐free medium and a 10% fetal bovine serum‐supplemented medium revealed that both the DNA synthesis and α5 subunit induction were independent of exogenous growth factors. The increased integrin α5b̃1 appears to function by reducing mitogenic activity since blockade of fibronectin binding to its receptor with a RGD peptide, a monoclonal anti‐fibronectin antibody, or a monoclonal anti‐α5 subunit antibody during the release from quiescence significantly stimulated DNA synthesis. On the other hand, stable overexpression of the α5 subunit resulted in decreased DNA synthesis. © 1995 Wiley‐Liss, Inc.
Cytoskeletal regulation of Caco‐2 intestinal monolayer paracellular permeability
Authors: Ma Thomas Y. , Hollander Daniel , Tran Le Thuy , Nguyen Don , Hoa Neil , Bhalla Deepak
Published: February 2005

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter‐grown Caco‐2 intestinal epithelial monolayers. Cytochalasin‐b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin‐b (5 m̈g/ml) and colchicine (2 × 10−5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin‐b‐induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco‐2 monolayer paracellular permeability. The cytochalasin‐b‐induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin‐D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4‐dinitrophenol or sodium azide). The cytochalasin‐b‐induced disturbance in Caco‐2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re‐tightening” following cytochalasin‐b removal was not affected by actinomycin‐D, cycloheximide, or colchicine, but was inhibited by 2,4‐dinitrophenol and sodium azide. The colchicine‐induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin‐b‐induced increase in Caco‐2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re‐tightening of paracellular pathways (following removal of cytochalasin‐b) resulted from energy‐mediated re‐assembly of pre‐existing actin microfilaments and intercellular junctional complexes. This re‐closure process did not require protein synthesis or microtubule‐mediated shuttling process. © 1995 Wiley‐Liss, Inc.
Two major pathways of zinc(II) acquisition by human placental syncytiotrophoblast
Authors: Bax Christopher M. R. , Bloxam David L.
Published: February 2005

Uptake of zinc into placental villous syncytiotrophoblast is the first step in its transfer from mother to fetus. To help characterise physiologically significant pathways of zinc accumulation by these cells, we incubated cultured layers of syncytiotrophoblast cells derived from human near‐term placental tissue with serum ultrafiltrate (containing the zinc complexed with low molecular mass serum constituents), dialysed serum (containing the zinc bound to the serum proteins) and whole serum, each of whose endogenous zinc was tracer‐labelled with 65Zn(II). Zinc label from both fractions of serum readily entered a rapidly labelled EDTA‐sensitive cellular compartment, probably representing zinc bound to the outside cell surface and in accumulative fashion, an EDTA‐resistant compartment, probably consisting largely of internalised cellular zinc. Movement of zinc into the EDTA‐resistant pool was strongly temperature‐dependent and did not occur via the EDTA‐sensitive pool from either serum source. Transfer of zinc from the low molecular mass serum fraction into the EDTA‐resistant pool was saturable, the concentration giving half‐maximal rate being 1.2 m̈mol/l nonprotein‐bound zinc. No nonsturable component was detected. Zinc from the serum protein‐bound fraction entered by a saturable component, already saturated at physiological total protein‐bound zinc concentration, and by an apparently nonsaturable component, not appreciably accounted for by nonspecific fluid‐phase endocytosis. The results show that zinc is acquired by placental syncytiotrophoblast from the low molecular mass serum zinc pool probably by a carrier‐mediated process, and at least as importantly, from the zinc bound to serum protein, possibly by an endocytic mechanism. © 1995 Wiley‐Liss, Inc. © 1995 Wiley‐Liss, Inc.
Members of the 70 kDa heat shock protein family specifically recognize sulfoglycolipids: Role in gamete recognition and mycoplasma‐related infertility
Authors: Boulanger J. , Faulds D. , Eddy E. M. , Lingwood C. A.
Published: February 2005

We have previously shown that several mycoplasma species associated with infertility bind specifically to sulfated glycolipids isolated from the mammalian reproductive tract. We now show that a germ cell‐specific sulfoglycolipid binding protein (SLIP 1), which is a potent inhibitor of sperm/egg binding in vitro, is immunologically related to the heat shock protein (Hsp) 70 family of stress proteins and that Hsps are surface antigens in male germ cells. Our present data demonstrate that several mycoplasma and mammalian Hsps share this glycolipid binding specificity in vitro, and suggest that surface Hsps can function as adhesins which mediate sulfoglycolipid recognition in infectious disease and normal reproductive physiology. © 1995 Wiley‐Liss Inc.
Protein kinase A regulates the degradation rate of Rs acetylcholine receptors
Authors: Xu Rufeng , Salpeter Miriam M.
Published: February 2005

Acetylcholine receptors at the neuromuscular junction of innervated vertebrate muscle (called Rs AChRs) have a stable degradation rate (t1/2∼8–12 days) which accelerates after denervation to a half‐life of ∼3 days, but can be restabilized by reinnervation or by cAMP. We examined the mechanism by which cAMP regulates the Rs degradation rate. When dibutyryl cAMP (DB‐cAMP) was applied to denervated mouse diaphragms in organ culture, it stabilized the accelerated degradation rate of the Rs. We found that this stabilization is reversible upon removal of the DB‐cAMP, is cAMP specific and is mediated by intracellular cAMP. A major observation of this study is that the cAMP‐induced stabilization of Rs AChRs is via protein kinase A (PKA), since H89, a PKA inhibitor, blocked the DB‐cAMP induced stabilization of Rs, and H85, an analog of H89, which does not inhibit PKA but does inhibit other kinases as efficiently as H89, did not prevent the DB‐cAMP‐induced stabilization of Rs degradation. These results suggest that the cAMP messenger system via a PKA‐dependent pathway could be among the mechanisms whereby the nerve regulates AChR degradation. © 1995 Wiley‐Liss Inc.
Significant induction of spermidine/spermine N1‐acetyltransferase without cytotoxicity by the growth‐supporting polyamine analogue 1,12‐dimethylspermine
Authors: Yang Jianing , Xiao Lei , Berkey Kimberly A. , Tamez Pamela A. , Coward James K. , Casero Robert A.
Published: February 2005

The superinduction of the polyamine catabolic enzyme spermidine/spermine N1‐acetyltransferase (SSAT) has been implicated in the cell type‐specific cytotoxic activity of some polyamine analogues. We now report that one polyamine analogue, 1, 12‐dimethylspermine (DMSpm), produces a large induction of SSAT with no significant effects on growth in the human large cell lung carcinoma line, NCI H157. This cell line has been demonstrated to respond to other analogues with SSAT superinduction and cell death. Treatment of the lung cancer cell line with DMSpm produces a rapid increase in SSAT activity and a near complete depletion of the natural polyamines. Additionally, DMSpm supports cell growth in cells which have been depleted of their natural polyamines by the ornithine decarboxylase inhibitor, 2‐difluoromethylornithine. The current results suggest that significant induction of SSAT can occur in the absence of cytotoxicity when the inducing polyamine analogue can support growth and that increased SSAT activity alone is not sufficient for cytotoxicity to occur. © 1995 Wiley‐Liss Inc.
Interleukin‐4 inhibits lipopolysaccharide‐induced expression of prostaglandin H synthase‐2 in human alveolar macrophages
Authors: Yano Tatsutoshi , Hopkins Harvey A. , Hemplel Stephen L. , Monick Martha , Hunninghake Gary W.
Published: February 2005

Several studies have shown that interleukin‐4 (IL‐4) down‐regulates synthesis of prostaglandin E2 (PGE2). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL‐4. LPS alone induced the synthesis of large amounts of PGE2 and prostaglandin H synthase‐2 (PGHS‐2) protein. This effect of LPS was suppressed by increasing amounts of IL‐4. Expression of LPS‐induced PGHS‐2 mRNA was also inhibited by IL‐4. In addition, IL‐4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS‐binding protein (LBP). We conclude that IL‐4 down‐regulates LPS‐induced release of PGE2, by reducing expression of the enzyme, PGHS‐2. One potential mechanism for this effect of IL‐4 is a reduced expression of CD14, which is the LPS‐LBP receptor. © 1995 Wiley‐Liss Inc.
Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways
Authors: Schnaper H. William , Barnathan Elliot S. , Mazar Andrew , Maheshwari Shailendra , Ellis Stefanie , Cortez Shirley L. , Baricos William H. , Kleinman Hynda K.
Published: February 2005

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue‐type plasminogen activator (tPA) and urokinase‐type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAl)‐1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady‐state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube‐forming activity. A fucosylated peptide comprising the amino‐terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA‐uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. © 1995 Wiley‐Liss Inc.
Stimulation of protein and DNA synthesis in mouse C2C12 satellite cells: Evidence for phospholipase d‐dependent and ‐independent pathways
Authors: Morrison Kenneth S. , Mackie Steven C. , Palmer Robert M. , Thompson Michael G.
Published: February 2005

In C2C12 myoblasts, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) stimulated a phospholipase D (PLD) to degrade phosphatidylcholine (PC) as measured by the release of choline and an increase in the formation of phosphatidic acid (PA) (or phosphatidylbutanol [PtdBuOH] in the presence of 0.5% butanol). Exogenous PLD also stimulated choline release, PA and PtdBuOH formation. The protein kinase C (PKC) inhibitor, Ro‐31‐8220, and PKC downregulation significantly inhibited the effects of TPA but Ro‐31‐8220 had no effect on PLD action. Neither basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF) increased PLD activity. All agonists stimulated protein synthesis during both a 90 min and a 6 hr incubation and increased RNA accretion after 6 hr. The response at 90 min was not inhibited by the transcription inhibitor, actinomycin D. Ro‐31‐8220 and PKC downregulation significantly inhibited all the effects of TPA. In contrast, Ro‐31‐8220 significantly inhibited the increase in RNA accretion elicited by PLD but had no effect on the ability of agonists other than TPA to enhance protein synthesis. All agonists also stimulated thymidine incorporation into DNA. The effects of EGF, bFGF, and PLD were rapid and transient whereas that of TPA was delayed and sustained. Ro‐31‐8220 and PKC downregulation significantly inhibited the response due to TPA. Furthermore, Ro‐31‐8220 also significantly inhibited the effects elicited by EGF and PLD but not that induced by bFGF. In differentiated myotubes, TPA and PLD, but not bFGF or EGF, again stimulated choline release and PtdBuOH formation. However, all agents failed to stimulate protein synthesis and RNA accretion. The data demonstrate the presence in C2C12 myoblasts, but not differentiated myotubes, of both a PLD‐dependent and PLD‐independent pathway(s) leading to the stimulation of protein synthesis, RNA accretion, and DNA synthesis. © 1995 Wiley‐Liss, Inc.
Melanoma cell spreading on fibronectin induced by 12(S)‐HETE involves both protein kinase C‐ and protein tyrosine kinase‐dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK)
Authors: Tang Dean G. , Tarrien Mark , Dobrzynski Philip , Honn Kenneth V.
Published: February 2005

Our previous work demonstrated that 12(S)‐HETE, a lipoxygenase metabolite of arachidonic acid, promoted B16 amelanotic melanoma (B16a) cell spreading on fibronectin. In the current study, we investigated the biochemical mechanisms of the 12(S)‐HETE induced response. 12(S)‐HETE treatment resulted in a time‐dependent increase in B16a cell spreading on fibronectin, which was blocked by either calphostin C or by genistein but not by H8. Two hours following cell plating, both spontaneous and 12(S)‐HETE promoted cell spreading reached their maximum (nearly 100%). Spontaneous cell spreading was inhibited by the select 12‐lipoxygenase inhibitor, BHPP, whose inhibitory effect could be overcome by increasing doses of exogenous 12(S)‐HETE. 12(S)‐HETE‐treated B16a cells plated on either fibronectin or cultured on their own extracellular matrix demonstrated increased vinculin and tyrosine‐phosphorylated proteins, which were colocalized at focal adhesions. The increase in vinculin localization to focal adhesions appeared to be a post‐transcriptional process, since 12(S)‐HETE treatment did not alter the overall protein level of vinculin in tumor cells, but resulted in a specific enrichment of vinculin to focal adhesions. Pretreatment of B16a cells with either calphostin C or genistein abolished 12(S)‐HETE‐increased formation of vinculin‐and phosphotyrosine‐containing focal adhesions. Immunoblotting using antiphosphotyrosine antibody 4G10 demonstrated, following 12(S)‐HETE stimulation, an increased tyrosine phosphorylation of several proteins in focal adhesions; most prominently, a ∼︁ 155 kd protein, a 120‐130 kd protein cluster, a 76 kd protein, and a 42/44 kd complex. Immunoprecipitation with anti‐phosphotyrosine antibody PY20 revealed increased tyrosine phosphorylation, post 12(S)‐HETE stimulation, of proteins migrating at 120, 76, and 42/44 kd, of which the 120 kd protein co‐migrated with pp125FAK. Immunoprecipitation with anti‐FAK antibody BC‐3 followed by immunoblotting with anti‐phosphotyrosine antibody RC20H demonstrated a time‐dependent hyperphosphorylation of pp125FAK. The present study suggests that 12(S)‐HETE promoted melanoma cell spreading on fibronectin involves tyrosine phosphorylation of pp125FAK and protein kinase C‐ and tyrosine kinase‐dependent focal adhesion formation. © 1995 Wiley‐Liss, Inc.
Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro
Authors: Allen Ronald E. , Sheehan Shannon M. , Taylor Richard G. , Kendall Teresa L. , Rice Glenna M.
Published: February 2005

The effect of hepatocyte growth factor (HGF) on the activation of quiescent rat skeletal muscle satellite cells was evaluated in vitro. Satellite cells from 9‐month‐old adult rats are quiescent in vivo and when cultured, display a protracted lag phase prior to division that is not present in satellite cells from neonatal or regenerating muscle. Under normal growth conditions, satellite cells divide for the first time between 42 and 60 hr. Hepatocyte growth factor increased proliferation in a dose‐dependent fashion prior to 48 hr with half‐maximal stimulation at approximately 3 ng/ml; in addition, heparin enhanced this activity. The time course of cyclin‐D1 and proliferating cell nuclear antigen (PCNA) expression was accelerated in HGF‐treated satellite cells, indicating that cells entered the cell cycle earlier. No significant effects on muscle‐derived fibroblast proliferation was observed. The signalling receptor for HGF is the product of the c‐met protooncogene, and rtPCR analysis of satellite cells 0–72 hr in culture demonstrated the presence of this message throughout this time period. The presence of c‐met in quiescent satellite cells, the ability of HGF to stimulate precocious entry into the cell cycle, and the previously described localization of HGF message in regenerating muscle (Jennische et al., 1993) indicate that HGF could act as an activator of quiescent satellite cells in vivo. © 1995 Wiley‐Liss, Inc.
Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture
Authors: Massey‐Harroche D. , Traverso V. , Mayran N. , Francou V. , Vandewalle A. , Maroux S.
Published: February 2005

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti‐annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non‐confluent or confluent cells which remained attached to the collagen‐coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen‐coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley‐Liss, Inc.
Characterization of transforming growth factor‐β growth regulatory effects and receptors on bovine mammary cells
Authors: Woodward T. L. , Dumont N. , O'Connor‐McCourt M. , Turner J. D. , Philip A.
Published: February 2005

Transforming growth factor‐β (TGF‐β) has been shown to inhibit mammary morphogenesis, growth, and differentiation in murine studies. We have characterized TGF‐β receptors and their autoregulation, and the growth response to TGF‐β1 and TGF‐β2 in cultured bovine mammary epithelium (MAC‐T) and fibroblasts. Affinity labelling studies revealed that fibroblast and epithelial cells contained type I, II, and III (betaglycan) receptors, with the type III receptor being the predominant binding component. On both fibroblasts and epithelial cells, TGF‐β1 and TGF‐β2 had equal binding affinities for the type I and II receptors, but TGF‐β2 had a higher affinity for the type III receptor. Also, preincubation of MAC‐T cells with 50 pM TGF‐β1 or TGF‐β2 markedly downregulated TGF‐β receptors. Proliferative response was measured using both total DNA and 3H‐thymidine incorporation. Both TGF‐β isoforms were effective in inhibiting proliferation of MAC‐T cells and fibroblasts. Inhibition of proliferation was not altered following immortalization of fibroblasts with SV‐40 Large‐T‐antigen (LT), even when the cells acquired a transformed phenotype. Inhibition of proliferation was not a result of cytotoxicity, as TGF‐β at concentrations 1,000‐fold higher than ED50 levels did not increase cell death. Moreover, the inhibition was reversible as shown by return of cellular proliferation to control levels following TGF‐β removal. Although growth inhibition was not transient as culture of MAC‐T cells in TGF‐β resulted in sustained inhibition of proliferation for at least 144 h. © 1995 Wiley‐Liss, Inc.
Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts
Authors: Carver Wayne , Molano Ivan , Reaves Titus A. , Borg Thomas K. , Terracio Louis
Published: February 2005

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF‐β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley‐Liss, Inc.
Fibroblast growth factor receptors 1 and 2 are differentially regulated in murine embryonal carcinoma cells and in response to fibroblast growth factor‐4
Authors: Ali Jahanara , Mansukhani Alka , Basilico Claudio
Published: February 2005

We have studied the expression of two of the receptors for fibroblast growth factors, FGFR‐1 and FGFR‐2, in response to ligand binding and in embryonal carcinoma (EC cells)> Exposure of mouse fibroblasts to FGF‐4 or FGF‐2 results in a drastic downregulation of the mRNA levels for FGFR‐2, while expression of FGER‐1 mRNA appears unaffected. Furthermore, FGF‐4 transformed cells display low levels of FGFR‐2 mRNA and these levels are significantly increased by treatment with anti FGF‐4 neutralizing antibodies. In undifferentiated F9 EC cells, the levels of FGFR‐2 mRNA are very low and increase substantially upon induction of differentiation. The level of mRNA for FGFR‐1 are again unaffected. To gain information on the regulation of expression of the gene encoding FGFR‐2 (bek) we have cloned the FGFR‐2 promoter region and used it to drive the expression of plasmids encoding the bacterial CAT enzyme. Transfection of these plasmids into FGF treated and untreated cells did not produce significant variation in CAT activity, suggesting that FGFR‐2 downregulation in response to ligand binding occurs mainly by a post‐transcriptional mechanism. In contrast, plasmids containing as little as 140 nt of the FGFR‐2 promoter region were regulated in F9 cells, showing substantially higher expression in differentiated than in undifferentiated cells. It appears therefore that FGFR‐2 expression in fibroblasts and EC cells is regulated by somewhat different mechanisms. In contrast, FGFR‐1 expression does not vary substantially under the conditions shown to affect FGFR‐2 expression. The implications of these findings are discussed. © 1995 Wiley‐Liss, Inc.
Growth control and gene expression in a new hepatocellular carcinoma cell line, Hep40: Inhibitory actions of vitamin K
Authors: Bouzahzah Boumediene , Nishikawa Yuji , Simon Daniela , Carr Brian I.
Published: February 2005

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF‐α, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF‐β family proteins. Affinity crosslinking gels using 125I‐labeled TGF‐β showed a significant decrease in the TGF‐β cell‐surface type II receptor in Hep40 cells, compared to the TGF‐β‐sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c‐myc, c‐jun, and prothrombin genes, in contrast to the actions of TGF‐β1 protein, which caused a decrease in the level of c‐myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF‐β with a unique TGF‐β receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF‐β as compared to vitamin K suggest that these two growth inhibitors act through differing pathways. © 1995 Wiley‐Liss Inc.
Plasma membrane‐dependent activation of gelatinase A in human vascular endothelial cells
Authors: Lewalle J. M. , Munaut C. , Pichot B. , Cataldo D. , Baramova E. , Foidart J. M.
Published: February 2005

The initiation of the angiogenic process requires a locally confined and time‐limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane‐dependent mechanism of activation has been described for several tumor and transformed cell lines. In the present study, we show that latent (72 kD) and mature (62‐59 kD) froms of gelatinase A are present in EC membrane fraction from Triton X‐114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62–59 kD mature forms. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane‐integrated MMP, the MT‐MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane‐dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis. © 1995 Wiley‐Liss Inc.