bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix Gla protein in vitro
Authors: Barone Leesa M. , Aronow Michael A. , Tassinari Melissa S. , Conlon Donna , Stein Gary S. , Lian Jane B. , Canalis Ernesto
Published: February 2005

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ‐carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin‐treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells
Authors: Andreatta‐Van Leyen Sheila , Hembree Joan R. , Eckert Richard L.
Published: February 2005

Insulin‐like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin‐like growth factor binding protein 3 (IGFBP‐3). In the present report we study the effects of epidermal growth factor (EGF) and all‐trans‐retinoic acid (RA) on IGFBP‐3 RNA and protein levels in human papillomavirus‐immortalized cervical epithelial cells. Treatment of ECE16‐1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP‐3 levels. In contrast, 1 μM RA increases IGFBP‐3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half‐maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP‐3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA‐dependent increase in IGFBP‐3 levels is dominant over the EGF suppression. The increased IGFBP‐3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP‐3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16‐D1, ECE16‐D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP‐3 levels and reducing the extracellular concentration of free insulin‐like growth factor I (IGFI) and/or, alternatively, IGFBP‐3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley‐Liss, Inc.
Role of laminin in the Morphogenetic cascade during Coculture of sertoli cells with peritubular cells
Authors: Tung Pierre S. , Fritz Irving B.
Published: February 2005

Observations summarized in this article demonstrate an essential role of laminin during the restructuring processes that occur during coculture of Sertoli cells with testicular peritubular cells. The data presented indicate that laminin becomes detectable on the free surfaces of Sertoli cells only after reaggregation of Sertoli cells begins, coincident with the initiation of repolarization at a specific stage of the morphogenetic cascade. We infer that laminin deposited at this time serves as a cohesion molecule that permits peritubular cells to come into close contact with Sertoli cells and subsequently to spread along the free surfaces of Sertoli cells. These conclusions and inferences are based on the following experiments. Cycloheximide‐treated peritubular cells in culture in MEM containing cycloheximide readily attach to laminin‐coated polystyrene surfaces. By contrast, added peritubular cells do not attach onto monolayers of Sertoli cells in monoculture or onto Sertoli cells plated on top of peritubular cells and maintained in coculture for periods of up to 48 h in cocultures maintained for 6 days, however, labeled peritbular cells readily adhere to the free surfaces of reaggregated Sertoli cells. Laminin, but not fibronectin, appears on the free surfaces of the reaggregated Sertoli cells atthis time, coinciding with the period of initial mound formation. The addition of antilaminin IgG, but not antifibronectin IgG, blocks the attachment of cycloheximide‐treated peritubular cells to laminin‐coated plates and also blocks the subsequent migration of peritubular cells required to form a monolayer. Similarly, anti‐laminin IgG inhibits the attachment and spreading of labeled peritubular cells seeded on the free surfaces of reaggregated Sertoli cells in mounds generated during the morphogenetic cascade. We interpret the combined data to indicate that the appearance of laminin on the free surfaces of Sertoli cells is required to permit peritubular cells to adhere and subsequently to migrate on Sertoli cell surfaces, resulting in the formation of a tubule‐like structure. © 1994 Wiley‐Liss, Inc.
Effect of glucose on pHin and [Ca2+]in in NIH‐3T3 cells transfected with the yeast P‐Type H+‐ATPase
Authors: Martínez Gloria M. , Martínez‐Zaguilán Raul , Gillies Robert J.
Published: February 2005

NIH‐3T3 cells transfected with yeast H+‐ATPases (RN1A cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334: 438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have alsoshown that RN1a cells are serum‐independent for growth, maintain a higher intracellular Ca2+(in), and glycolyze more rapidly than their non‐transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87: 7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2: 159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non‐transformed counterparts, NIH‐3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cell is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH‐3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH‐3T3 cells. Glucose addition to NIH‐3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca2+‐ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP‐depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump. © 1994 Wiley‐Liss, Inc.
Role of intracellular Ca2+ stores in the regulation of electrogenic plasma membrane Ca2+ uptake in a B‐lymphocytic cell line
Authors: Marriott Ian , Bost Kenneth L. , Mason Michael J.
Published: February 2005

Experiments were undertaken to investigate the role of intracellular Ca2+ stores in the regulation of Ca2+ uptake in the cultured B‐lymphocytic cell line CH12.LX.C4.5F5. Release of intracellular Ca2+ stores by addition of thapsigargin was accompanied by a biphasic increase in intracellular calcium concentration ([Ca2+]i). The initial rise in [Ca2+]i was due to release of Ca2+ from intracellular stores as determined by its maintenance in the absence of extracellular Ca2+. The secondary phase was (1) dependent on the presence of extracellular Ca2+, (2) inhibited by 5 mM extracellular Ni2+, and (3) inhibited by high K+, consistent with electrogenic Ca2+ uptake from the extracellular medium. In order to more accurate y investigate the electrogenic nature of this pathway we measured the membrane potential changes accompanying Ca2+ influx stimulated by release of Ca2+ from intracellular stores using bis(1,3‐diethylthiobarbituric acid trimethine) oxonol in Bapta‐loaded cells. Addition of 5 mM Ca2+ to cells pretreated with doses of thapsigargin or ionomycin shown to release intracellular Ca2+ stores induced a depolarization which was (1) dependent upon extracellular Ca2+, (2) abolished by 5 mM Ni2+, (3) independent of extracellular Na+, and (4) dependent upon Bapta loading. This depolarization was followed by a charybdotoxin‐sensitive repolarization consistent with secondary activation of K+ channels. Changes in [Ca2+]i monitored under identical conditions were monitored fluorimetrically using indo‐1 and were found to correlate with the changes in Em. On the basis of these data we conclude that an electrogenic Ca2+ ‐permeable pathway exists in this B‐lymphocytic cell line which is regulated by the degree of filling of an internal Ca2+ ‐store. © 1994 Wiley‐Liss, Inc.
Uptake of biotin by human hepatoma cell line, Hep G2: A carrier‐mediated process similar to that of normal liver
Authors: Said Hamid M. , Ma Thomas Y. , Kamanna Vaijinath S.
Published: February 2005

Little is known about the cellular and molecular regulation of the uptake process of the water‐soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human‐derived liver cells, Hep G2. Uptake to biotin by Hep G2 cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na+ gradient‐dependent as indicated by studies of Na+ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of 3H‐biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G2 cells with sulfhydryl group inhibitors (e.g., p‐chloromer‐curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G2 cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na+ uptake in Hep G2 cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na+‐gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G2 cells for in‐depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley‐Liss, Inc.

This article is a US Government work, and as such, is in the public domain in the United State of America

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In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: A juxtacrine model for migration and adhesion of osteoclast precursors
Authors: Formigli Lucia , Orlandini Sandra Zecchi , Benvenuti Susanna , Masi Laura , Pinto Antonio , Gattel Valter , Bernabei Pietro Antonio , Robey Pamela Gehron , Collin‐Osdoby Patricia , Brandi Maria Luisa
Published: February 2005

The role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone‐derived endothelial and preosteoclastic cells. Insulin‐like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell‐specific and mediated through the binding of vitronectin and fibronection receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny. © 1995 Wiley‐Liss, Inc.
Relationship between actions of transforming growth factor (TGF)‐β and cell surface expression of its receptors in clonal osteoblastic cells
Authors: Takeuchi Yasuhiro , Fukumoto Seiji , Matsumoto Toshio
Published: February 2005

Various osteoblastic cell lines were examined for the relationship between the presence of cell‐surface transforming growth factor (TGF)‐β receptors and the synthesis of matrix proteins with their responsiveness to TGF‐β. Treatment with TGF‐β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3‐E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF‐β1 markedly stimulated the synthesis of decorin in MC3T3‐E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF‐β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF‐β1 enhanced fibronectin synthesis. TGF‐β1 did not show any of these effects in UMR 106 cells. Receptor cross‐linking studies revealed that only MC3T3‐E1 and MG 63 cells had both types I and II signal‐transducing receptors for TGF‐β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3‐E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF‐β1 under these conditions. UMR 106 cells with no response to TGF‐β1 had neither of the signal‐transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF‐β1, that the responsiveness of osteoblastic cells to TGF‐β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF‐β1 on fibronectin synthesis can develop with little binding of TGF‐β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell‐surface receptors for TGF‐β determines the responsiveness of osteoblastic cells to TGF‐β and that changes in cell‐surface TGF‐β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley‐Liss, Inc.
Reciprocal induction of tumor necrosis factor‐α and interleukin‐β activity mediates fibronectin synthesis in coronary artery smooth muscle cells
Authors: Molossi Silvana , Clausell Nadine , Rabinovitch Marlene
Published: February 2005

We previously demonstrated an immune‐inflammatory response associated with increased expression of interleukin (IL)‐β and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL‐β was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co‐induction of IL‐β and tumor necrosis factor (TNF)‐α has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF‐α expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]‐methionine radiolabeling and gelatin‐sepharose extraction. As previously shown with IL‐β, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF‐α antibodies, and exogenous TNF‐α upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF‐α‐stimulated fibronectin production was downregulated to or below control levels in the presence of IL‐β antibodies. Likewise, IL‐β‐stimulated fibronectin synthesis was downregulated to control levels when TNF‐α neutralizing antibodies were added. Combining TNF‐α and IL‐β enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL‐β and TNF‐α activity and provide the first demonstration of a ‘cytokine loop’ modulating matrix production. © 1995 Wiley‐Liss, Inc.
Volume regulation in leukocytes: Requirement for an intact cytoskeleton
Authors: Downey Gregory P. , Grinstein Sergio , Sue‐A‐Quan Andrea , Czaban Barbara , Chan Chi Kin
Published: February 2005

Animal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL‐60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL‐60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F‐actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F‐actin content of the different cells was determined by NBD‐phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 ± 0.01 for CB and 0.48 ± 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL‐60 cells or human neutrophils. To study the effects of an increase in F‐actin on volume regulation, neutrophils were treated with the chemoattractant f‐Met‐Leu‐Phe or with an antibody (Ab) to β2 integrins followed by a cross‐linking secondary Ab. Despite an increase in F‐actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation. © 1995 Wiley‐Liss, Inc.
Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line
Authors: Delezay Olivier , Verrier Bernard , Mabrouk Kamel , van Rietschoten Jurphaas , Fantini Jacques , Mauchamp Jean , Gerard Corinne
Published: February 2005

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT‐29‐D4. Isotopic tracer flux measurements show that differentiated HT‐29‐D4 cells possess a sodium‐dependent α‐methyl‐D‐glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D‐glucose, D‐galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium‐dependent AMG transporter with a Km of 1.2 ± 0.12 mM and a Vmax of 3.24 ± 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 ± 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402‐Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT‐29‐D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT‐29‐D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference. © 1995 Wiley‐Liss, Inc.
Intraphagosomal oxygen in stimulated macrophages
Authors: James Philip E. , Grinberg Oleg Y. , Michaels George , Swartz Harold M.
Published: February 2005

A new electron paramagnetic resonance (EPR)‐based method was developed to obtain selective information on pO2 in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen‐sensitive probe (4‐(Trimethylammonium) 2,2,6,6‐tetramethylpiperidine‐d17‐1‐oxyl iodide (d‐Cat1)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐d16‐1‐oxyl (15N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley‐Liss, Inc.
Mechanical stimulation of skeletal muscle increases prostaglandin F2α production, cyclooxygenase activity, and cell growth by a pertussis toxin sensitive mechanism
Authors: Vandenburgh Herman H. , Shansky Janet , Solerssi Rosa , Chromiak Joseph
Published: February 2005

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long‐term production of prostaglandin F2α, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P <.005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch‐activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's Km, with little change in its Vmax. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen‐inducible isoform of cyclooxygenase (PGHS‐2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch‐induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch‐induced cyclooxygenase activation. Pertussis toxin also inhibited stretch‐induced increases in PGF2α production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein‐dependent process involving the activation of cyclooxygenase, an immediate early gene product. © 1995 Wiley‐Liss, Inc.
Evidence that cell survival is controlled by interleukin‐3 independently of cell proliferation
Authors: Berridge Michael V. , Horsfield Julie A. , Tan An S.
Published: February 2005

Hemopoietic cell proliferation is controlled by a set of polypeptide growth factors and regulatory molecules that bind to cell surface receptors inducing cellular responses. Maintenance of a viable state, cell growth, DNA synthesis and mitosis are basic properties of proliferating cells, but links between growth factor receptors and each of these cellular outcomes are poorly understood. Most studies have monitored DNA synthesis as a measure of progression through the cell cycle or directly measured viable cell numbers, but cell survival per se as an output of receptor activation by ligand, has received little attention. In this study we have used a bone marrow‐derived murine cell line that is dependent on interleukin‐3 for growth, to investigate the relationship between DNA synthesis and a biochemical marker of cell survival, reduction of the tetrazolium salt, MTT. We show that at times up to 6 hr, continued DNA synthesis, RNA synthesis, protein synthesis, and mitochondrial respiration are not necessary for background or IL‐3‐stimulated MTT reduction. Furthermore, dibutyryl cyclic AMP promoted background and IL‐3‐dependent MTT reduction while simultaneously inhibiting DNA synthesis. These results provide evidence that IL‐3 controls events involved in MTT reduction and cell survival independently of DNA synthesis. © 1995 Wiley‐Liss, Inc.
De novo synthesis of glutathione is required for both entry into and progression through the cell cycle
Authors: Poot Martin , Teubert Heidi , Rabinovitch Peter S. , Kavanagh Terrance J.
Published: February 2005

To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH‐3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose‐dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO‐induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit. © 1995 Wiley‐Liss, Inc.
Mitogenic activity of neu differentiation factor/heregulin mimics that of epidermal growth factor and insulin‐like growth factor‐I in human mammary epithelial cells
Authors: Ram Tracy G. , Kokeny Kristine E. , Dilts Cheryl A. , Ethier Stephen P.
Published: February 2005

Recently, a family of growth factors has been described that activates erbB‐2 receptors. These factors, known as the neu differentiation factors (NDF) or heregulins (HRG), induce tyrosine phosphorylation of erbB‐2 receptors as a result of their direct interaction with either erbB‐3 or erbB‐4 receptors. Although it is known that expression of erbB‐2 receptors has relevance in human breast cancer progression, how erbB‐2, ‐3 and ‐4 receptors regulate mammary epithelial cell proliferation is not known. Therefore, experiments were carried out to study the mitogenic activity of NDF/HRG on the human mammary epithelial cell line MCF‐10A which can be cultured continuously under serum‐free conditions. MCF‐10A cells, like primary cultures of normal human mammary epithelial cells, express an absolute requirement for exogenous epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) for growth. The results of these experiments indicate that NDF/HRG can induce tyrosine phosphorylation of p185erbB‐2 in MCF‐10A cells and is mitogenic for these cells. This is consistent with the coexpression of erbB‐2 and erbB‐3 mRNA that we have observed in MCF‐10A cells. In addition, we found that NDF/HRG can substitute for either EGF or IGF‐I to stimulate proliferation of these cells. The ability to substitute for both EGF and IGF‐I is a unique property of NDF/HRG and is not shared by other members of the EGF or IGF family of growth factors, nor by other factors that we have studied. A striking isoform specificity was also observed which indicated that the β‐isoforms of NDF/HRG were greater than ten times more mitogenic than the α‐isoforms. We also examined the mitogenic activity of NDF/HRG on MCF‐10A cells that overexpress the erbB‐2 receptor as a result of infection with a retroviral vector containing the human c‐erbB‐2 gene (MCF‐10AerbB‐2 cells). These studies indicated that MCF‐10AerbB‐2 cells have increased sensitivity to the mitogenic effects of NDF/HRG and that these cells are responsive to the α‐isoforms of NDF/HRG at physiological concentrations. Thus, NDF/HRG is a dual specificity growth factor for human mammary epithelial cells, and the responsiveness of the cells to NDF/HRG is influenced by the level of expression of erbB‐2 receptors. © 1995 Wiley‐Liss, Inc.
Repression of tissue inhibitor of matrix metalloproteinase expression by all‐trans‐retinoic acid in rat bone cell populations: Comparison with transforming growth factor‐βT1
Authors: Overall Christopher M.
Published: February 2005

Retinoids and transforming growth factor‐b̃1 (TGF‐b̃1) reduce the transcriptional activation of matrix metalloproteinases (MMPs) and increase the expression of the specific tissue inhibitor of MMPs (TIMP‐1) in fibroblasts. In contrast, all‐trans‐retinoic acid (retinoic acid) increases MMP expression in osteoblasts. Therefore, the mechanistic aspects of TIMP‐1 regulation by retinoic acid in primary cultures of rat calvarial bone cell populations were studied and compared with those of TGF‐b̃1 to determine if modulation of TIMP‐1 would augment MMP expression. Retionic acid was found to reduce TIMP‐1 mRNA levels after 24 and 72 hr of culture by up to 60% in a dose‐dependent manner. Maximal inhibition occurred at 10−6 M retinoic acid with half maximal repression at ∼5 × 10−8 M. To determine the half life of TIMP‐1 mRNA, the specific RNA polymerase II inhibitor DRB was added to cultures and the chase RNA analyzed by slot blots. TIMP‐1 mRNA had a half life of ∼14 hr and this was unaltered by retinoic acid treatment, suggesting that retinoic acid exerts its effects on TIMP‐1 transcriptionally. When retinoic acid was added to cycloheximide‐treated cultures TIMP‐1 mRNA levels were reduced at 5 hr compared with controls. This showed that ongoing protein synthesis was not required to mediate the retinoic acid repression of TIMP‐1 mRNA levels and supports the evidence that retinoic acid acts at the transcriptional level to reduce TIMP‐1 expression. In contrast, TGF‐b̃1 increased TIMP‐1 mRNA levels by 3.5‐fold at 24 hr to>10‐fold at 72 hr without alterations in mRNA stability indicating that transforming growth factor (TGF)‐b̃1 also acts at the transcriptional level to upregulate TIMP‐1 expression in bone cells. Thus, these studies have revealed that TIMP‐1 regulation by retinoic acid is different in osteoblasts from other cells and that retinoic acid has the property of generating resorptive and formative cell phenotypes in a tissue‐specific manner. In bone, reduced TIMP‐1 expression would favor bone matrix degradation and bone resorption that is a characteristic action of retinoids. © 1995 Wiley‐Liss, Inc.
Genetic factors influencing murine hematopoietic productivity in culture
Authors: Phillips Robert L. , Couzens Matthew S. , Van Zant Gary
Published: February 2005

In order to study a previously described genetic difference manifested in stem cell kinetics of specific mouse strains, effects of this putative gene, stk, were measured on growth and expansion of stem and progenitor cell populations ex vivo. Bone marrow cells from each of two inbred mouse strains, C57BL/6J and DBA/2J, were placed into separate bioreactor cultures perfused continuously with growth medium containing erythropoietin (Epo), interleukin‐3 (IL‐3), granulocyte‐macrphage colony stimulating factor (GM‐CSF), and Kit ligand as well as 5% CO2. Expansion of cell numbers reached 20‐fold for DBA/2J and 10‐fold for C57BL/6J marrow within about 1 week of culture. Significant production was also seen of colonyforming unit (CFU)‐GM (up nine‐fold from input levels) just prior to the cell production peak, and, importantly, moderate expansion of day 12 colony‐forming unit‐spleen (CFU‐S; two‐ to threefold) occurred as well, although CFU‐S production peaked at a relatively short 4 days. CFU‐S and CFU‐GM levels declined rapidly in culture, either because of unfavorable growth conditions or terminal differentiation. Attempts to remove toxic metabolites by increasing the media perfusion rate resulted in a boost in cell expansion capability by DBA/2J marrow. In bioreactors in which stromal cells were established before marrow inoculation, there was greater expansion of CFU‐S (especially by DBA/2J) and CFU‐GM, although total cell yield appeared to be unaffected, perhaps because the maximum cell density had already been reached. The relative high potential for CFU‐S expansion measured in DBA/2J marrow over that of C57BL/6J will be useful in following genetic contributions to bone marrow production capacity. © 1995 Wiley‐Liss, Inc.
Insulin‐like growth factor I receptor is required for the mitogenic and transforming activities of the platelet‐derived growth factor receptor
Authors: Deangelis Tiziana , Ferber Andres , Baserga Renato
Published: February 2005

R− cells are 3T3‐like cells derived from mouse embryos in which the insulin‐like growth factor I (IGF‐I) receptor (IGF‐IR) genes have been disrupted by targeted homologous recombination. These cells cannot grow in serum‐free medium supplemented by the growth factors that sustain the growth of other 3T3 cell lines, and cannot be transformed by oncogenes that easily transform wild type mouse embryo cells. We have used these cells to study the role of the IGF‐IR in the growth and transformation of cells overexpressing the platelet‐derived growth factor (PDGF)‐b̃b̃ receptor. We report that an overexpressed PDGF‐b̃b̃ receptor fails to induce mitogenesis or transformation in cells lacking the IGF‐IR, while capable of doing so in cells expressing the IGF‐IR. We conclude that the ability of the activated PDGF‐b̃b̃ receptor to stimulate cell proliferation and transformation requires a funcitional IGF‐IR. © 1995 Wiley‐Liss, Inc.
Macrophage colony‐stimulating factor induces thrombospondin I production by cultured human macrophages
Authors: De Nichilo Mark O. , Burns Gordon F.
Published: February 2005

The role of colony‐stimulating factors (CSFs) in regulating the synthesis of thrombospondin 1 (TSP1) by cultured human macrophages is investigated. Macrophage (M)‐CSF is shown rapidly and transiently to induce two predominant species of TSP1 mRNA. One of these species was 3.2 kb in size and appeared to be specific to M‐CSF‐stimulated macrophages. Adherent M‐CSF‐treated macrophages are also shown to express abundant surface cell‐associated TSP rapidly when examined by indirect immunofluorescence staining. Granulocyte‐macrophage (GM)‐CSF induced TSP1 mRNA at a later time point, and this was attributable to the effects of endogenous M‐CSF induced by the GM‐CSF; the GM‐CSF‐treated cells did not display surface‐associated TSP after 3 hr of treatment. Analysis of the TSP1 protein synthesised by the M‐CSF‐treated macrophages revealed the expected trimeric form of the molecule. In addition, an unidentified 95‐kDa protein was found to be covalently associated with immunoreactive TSP1, and this appeared to be specific to the macrophages as it was not found in TSP1 precipitated from other cell types. It is suggested that the induction of TSP1 by M‐CSF may play an important role in the major physiological functions of macrophages. © 1995 Wiley‐Liss, Inc.
Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture
Authors: Ågren Ulla M. , Tammi Markku , Tammi Raija
Published: February 2005

We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three‐dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with 3H‐glucosamine and 3H‐thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan‐link protein complex. A low concentration of HC (10−9 M) stimulated the incorporation of 3H‐glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5‐day culture in 10−9 M HC. On the other hand, a high concentration of HC (10−5 M) reduced both synthesis (‐42%) and degradation (‐46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5‐day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10−5 M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10−5 M) concentration of HC was effective, inhibiting the incorporation of 3H‐glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers. © 1995 Wiley‐Liss, Inc.
Competition between c‐fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3‐E1 osteoblastic cells
Authors: Kuroki Yasuo , Shiozawa Shunichi , Kano Junichi , Chihara Kazuo
Published: February 2005

Interaction between c‐fos and 1,25(OH)2 vitamin D3 (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3‐E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c‐fos transfectants that overexpressed c‐fos gene to a comparable level as those of the control transfectants. The gel shift assay showed that vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c‐fos gene, suggesting that c‐fos gene product, c‐Fos, may inhibit the binding of VDR complex to VDRE by making a c‐Fos‐VDR complex. The result suggests the existence of a fine tuning between c‐fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion. © 1995 Wiley‐Liss, Inc.
Laminarin sulfate mimics the effects of heparin on smooth muscle cell proliferation and basic fibroblast growth factor‐receptor binding and mitogenic activity
Authors: Miao Hua‐Quan , Ishai‐Michaeli Rivka , Peretz Tamar , Vlodavsky Israel
Published: February 2005

Heparin and heparin‐like molecules may function, apart from their effect on hemostasis, as regulators of cell growth and neovascularization. We investigated whether similar effects are exerted by laminarin sulfate, an unrelated polysulfated saccharide isolated from the cell wall of seaweed and composed of chemically O‐sulfated b̃‐(1,3)‐linked glucose residues. Laminarin sulfate exhibits about 30% of the anticoagulant activity of heparin and is effective therapeutically in the prevention and treatment of cerebrovascular diseases. We characterized the effect of laminarin sulfate on interaction of the heparin‐binding angiogenic factor, basic fibroblast growth factor (bFGF), with a naturally produced subendothelial extracellular matrix (ECM) and with cell surface receptor sites. Laminarin sulfate (1‐2 m̈g/ml) inhibited the binding of bFGF to ECM and to the surface of vascular smooth muscle cells (SMC) in a manner similar to that observed with heparin. Likewise, laminarin sulfate efficiently displaced both ECM‐and cell‐bound bFGF at concentrations as low as 1 m̈g/ml. Both laminarin sulfate and heparin efficiently induced restoration of bFGF receptor binding in xylosyltransferase‐deficient CHO cell mutants defective in initiation of glycosaminoglycan synthesis. Moreover, laminarin sulfate elicited bFGF receptor activation and mitogenic response in heparan sulfate(HS)‐deficient, cytokine‐dependent lymphoid cells. These results indicate that laminarin sulfate effectively replaced the need for heparin and HS in the induction of bFGF receptor binding and signaling. In other experiments, laminarin sulfate was found to inhibit the proliferation of vascular SMC in a manner similar to that observed with heparin. These effects of laminarin sulfate may have potential clinical applications in diverse situations such as wound healing, angiogenesis, and atherosclerosis. © 1995 Wiley‐Liss, Inc.
Induction of angiogenesis by smooth muscle cell‐derived factor: Possible role in neovascularization in atherosclerotic plaque
Authors: Kuzuya Masafumi , Satake Shosuke , Esaki Teiji , Yamada Kazuyoshi , Hayashi Toshio , Naito Michitaka , Asai Kanichi , Iguchi Akihisa
Published: February 2005

The development of atherosclerotic plaque is associated with neovascularization in the thickened intima and media of vascular walls. Neovascularization may have a role in the progression of atherosclerotic plaque as well as in the development of intraplaque hemorrhage. However, the mechanism and stimulus for neovascularization in atherosclerotic plaque are unknown. We postulated that smooth muscle cells (SMCs), a major cellular component in the vascular wall, might contribute to the induction of neovascularization in atherosclerotic plaque through the secretion of an angiogenic factor. We observed that endothelial cells (ECs) cultured on collagen gel with SMC‐conditioned medium became spindle shaped, invaded the underlying collagen gel, and organized a capillary‐like branching cord structure in the collagen gel. The conditioned medium also stimulated EC proliferation and increased the EC‐associated plasminogen activator activity. The angiogenic factor in SMC‐conditioned medium was retained in a heparin‐Sepharose column and eluted with 0.9 M NaCl. Neutralizing anti‐vascullar endothelial growth factor (VEGF) antibody attenuated the angiogenic activity in the conditioned medium, including the induction of morphologic changes in ECs, mitogenic activity, and increased plasminogen activator activity associated with ECs. Immunoblotting analysis confirmed the secretion of VEGF from SMCs. These observations indicate that SMC may be responsible for the neovascularization in atherosclerotic plaque through the secretion of VEGF. © 1995 Wiley‐Liss, Inc.
Insulin‐like growth factor‐1 stimulates amino acid uptake by the cultured human placental trophoblast
Authors: Karl Peter I.
Published: February 2005

Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory characterized the uptake of α‐aminoisobutyric acid (AIB) stimulated by insulin in the cultured human placental trophoblast The current study evaluated the effect of insulin‐like growth factor‐1 (IGF‐1) on AIB uptake in cultured human placental trophoblasts. Na+‐dependent AIB uptake was significantly stimulated by IGF‐l in a time‐dependent manner, as early as 30 min after hormone exposure. The maximum effect was at 2–4 hr of continuous exposure to IGF‐l and the stimulation was dependent upon IGF‐1 concentration approaching maximal stimulation at 50 ng.ml−1. AIB uptake was inhibited by increasing concentrations of α‐(methylamino)isobtyric acid (MeAIB). Approximately 75% of basal (unstimulated) Na+‐dependent AIB uptake was inhibited by MeAIB. The IGF‐1‐stimulated increment above basal AIB uptake was completely inhibited by MeAIB. IGF‐1 increased the maximum uptake yelocity but not Km. Using equimolar concentrations, stimulation was greater with IGF‐1 then with IGF‐2. Stimulation by IGF‐1, but not insulin, was inhibited by anit‐IGF‐1 receptor antibody, indicating mediation via the IGF‐1 receptor. H7, a nonspecific inhibitor of serine‐threonine kinase, inhibited IGF‐1‐dependent stimulation of AIB uptake. In addition, calphostin C (a specific inhibitor of protein kinase C), but not H89 (a specific inhibitor of protein kinase A), inhibited the IGF‐1 action. This study further characterizes regulated amino acid uptake by the human placental trophoblasts and demonstrates that the Na+‐dependent component of AIB uptake is stimulated by physiologic concentrations of IGF‐1. © 1995 Wiley‐Liss Inc.