bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of PDGF receptor tyrosine kinase activity from PDGF‐mediated inhibition of gap junctional communication
Authors: Pelletier Diane B. , Boynton Alton L.
Published: February 2005

Gap junction‐mediated intercellular communication (GJC) may play an important role in cell proliferation and transformation since GJC is inhibited by growth factors, oncogenes, tumor promoters, and carcinogens. We have studied inhibition of GJC by platelet‐derived growth factor‐BB (PDGF) in the mouse fibroblast cell line C3H/10T1/2 and have sought to determine whether PDGF‐induced inhibition of GJC is mediated by the PDGF receptor tyrosine kinase (RTK). PDGF‐mediated inhibition of GJC was rapid and transient, with maximal inhibition occurring 40 min after PDGF addition and GJC returning to control levels after 70 min. The effect of PDGF on GJC was concentration‐dependent, with maximal inhibition of 90% or greater occurring at 10 ng/ml PDGF. Stimulation of RTK activity, as determined by antiphosphotyrosine immunoblot analysis of PDGF receptor and the receptor substrates phospholipase C‐γl (PLC‐γl) and guanosine triphosphatase activating protein (GAP), was also concentration‐dependent. Inhibition of GJC required a greater concentration of PDGF than did stimulation of RTK activity. The tyrosine kinase inhibitor genistein blocked PDGF‐induced RTK activity, as measured by PDGF receptor, PLC‐γl, and GAP tyrosine phosphorylation, in a concentration‐dependent manner but did not affect PDGF‐mediated inhibition of GJC. Genistein alone had no effect on GJC or PDGF receptor expression. PDGF treatment in the presence or absence of genistein resulted in phosphorylation of the connexin 43 protein on nontyrosine residues. These results suggest that inhibition of GJC by ligand‐activated PDGF receptor is dissociable from the RTK activity responsible for PDGF, PLC‐γl, and GAP phosphorylation. © 1994 Wiley‐Liss, Inc.
Modulation of thrombospondin expression during differentiation of embryonal carcinoma cells
Authors: Liska Deann J. , Hawkins Richard , Wikstrom Kristina , Bornstein Paul
Published: February 2005

The thrombospondins (TSPs) are a family of extracellular glycoproteins that display distinct patterns of temporal and spatial expression during development. In this study, we investigated the expression of two of the TSPs–TPS1 and TSP2– during the course of differentiation of embryonal carcinoma cells in vitro. We report that both TSP1 and TSP2 mRNA and protein synthesis are induced during the differentiation of P19EC cells into neurons, glial cells, and fibroblasts. Immunofluorescence studies indicate that TSP1 displays a fibrillar pattern of staining, characteristic of an extracellular matrix protein, in differentiated P19EC cells. In contrast, TSP2 is cell‐associated and is present on differentiated P19EC cells and on primary neurons and glial cells obtained from a 17‐day embyronic mouse cerebral cortex. Interestingly, although both TSP1 and TSP2 are more prevalent in areas of differentiated cells, they display distinct patterns of deposition. These observations suggest that TSP1 and TSP2 may function differently during neurogenesis. The response of TSP1 and TSP2 to differentiation of P19EC cells indicates that this cell system will serve as a valuable model for the study of TSP expression and function during neurogenesis. © 1994 Wiley‐Liss, Inc.
Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC‐PK1 cells
Authors: Yet Shaw‐Fang , Kong Cheng‐Te , Peng Hua , Lever Julia E.
Published: February 2005

The porcine kidney epithelial cell line LLC‐PK1 expresses a sodium‐coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and γ‐glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers–trehalase and γ‐glutamyltranspeptidase. By contrast, trehalase and γ‐glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers. © 1994 Wiley‐Liss, Inc.
Ligation of CD3 triggers transmembrane proximity between LFA‐1 and cortical microfilaments in a cytotoxic T cell clone derived from tumor infiltrating lymphocytes: A quantitative resonance energy transfer microscopy study
Authors: Poo Haryoung , Fox Bernard A. , Petty Howard R.
Published: February 2005

We have explored the transmembrane associations of leukocyte function associated antigen‐1 (LFA‐1) in response to T cell receptor ligation using resonance energy transfer (r.e.t.) microscopy to detect receptor to microfilament proximity. R.e.t. was detected using both imaging and photon counting techniques. T cells were labeled with fluorescein‐conjugated F(ab')2 fragments of an anti—LFA‐1 monoclonal antibody. Cells were incubated at 37°C on unmodified glass surfaces and surfaces coated with anti‐CD3 or anti—H‐9 antibodies. Microfilaments of fixed cells were labeled with rhodamine‐phalloidin. R.e.t. was not affected on unmodified (blank) or irrelevant antibody‐treated (H‐9) surfaces. However, both fluorescence images and photon count rates were significantly enhanced when cells bound to anti‐CD3‐coated surfaces. This enhancement was not due to a general effect of T cell activation on transmembrane cytoskeletal proximity since CD45‐phalloidin r.e.t. was not affected by CD3 ligation. These experiments provide direct physical evidence that ligation of the CD3 complex specifically increases the proximity of LFA‐1 and microfilaments, which may be relevant to T cell mediated adherence reactions. © 1994 wiley‐Liss, Inc.
Growth modulation and proteoglycan turnover in cultured mesangial cells
Authors: Wang Aimin , Fan Mei‐Ying , Templeton Douglas M.
Published: February 2005

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin‐like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with 35SO42− by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (Kd of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (Kd = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with Kd values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (Kd = 0.17) was recovered from the medium. After 16 h of labeling in serum‐free medium, about 60% of macromolecular 35S was cell‐associated and 40% was in the medium. Cell‐associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [3H]‐thymidine into DNA after release of serum‐starved RMC from growth arrest. Heparin acted prior to the G1/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley‐Liss, Inc.
Serum response elements mediate protein kinase C dependent transcriptional induction of early growth response gene‐1 by arginine vasopressin in rat mesangial cells
Authors: Rupprecht Harald D. , Sukhatme Vikas P. , Rupprecht Anneli P. , Sterzel R. Bernd , Coleman David L.
Published: February 2005

Arginine vasopressin (AVP) regulates glomerular hemodynamics, alters extracellular matrix production, and induces proliferation of glomerular mesangial cells (MCs). Therefore, AVP may play a role in glomerular sclerosis and the progression of chronic renal failure. To investigate changes in early gene expression which may link intracellular biochemical events with changes in MC phenotype following AVP stimulation, we studied expression of the Early growth response gene‐1 (Egr‐1). Nuclear run off assays demonstrate that AVP induces Egr‐1 at the tran‐scriptional level. Transcriptional induction was, like induction of mitogenesis, dependent upon activation of protein kinase C (PK C). Promoter deletion analysis revealed that the region critical for Egr‐1 inducibility by AVP contained several serum response element (SRE) consensus sequences. Sequential deletion of these SREs led to a drop in AVP‐stimulated promoter activity. AVP was also able to stimulate transcription from a construct containing an Egr‐1 SRE upstream of a heterologous promoter and this effect required activation of PK C. Electrophoretic mobility shift assays, using an Egr‐1 SRE as probe, demonstrate up to four protein‐SRE complexes of differing size that undergo modest quantitative changes following AVP stimulation. These data in MCs suggest that upstream SREs mediate transcriptional induction of Egr‐1 by AVP in a PK C‐dependent fashion and that changes in DNA‐protein interaction involving the SREs may be in part responsible for this effect. © 1994 wiley‐Liss, Inc.
Inhibition of the chondrocyte phenotype by retinoic acid involves upregulation of metalloprotease genes independent of TGF‐β
Authors: Ballock R. Tracy , Heydemann Ahlke , Wakefield Lalage M. , Flanders Kathleen C. , Roberts Anita B. , Sporn Michael B.
Published: February 2005

Retinoic acid has been identified as a key morphogen governing pattern formation in the developing cartilaginous skeleton. Retinoids have also been implicated in the premature closure of the cartilage growth plate following vitamin A intoxication or administration of retinoids for dermatologic conditions. Previous studies of the mechanism of action of retinoids in non‐chondrogenic cells have concluded that retinoic acid is a negative regulator of AP‐1 responsive metalloprotease genes. We show that inhibition of expression of the cartilage phenotype by retinoic acid in epiphyseal chondrocytes is associated with positive regulation of AP‐1 responsive metalloprotease genes, as well as induction of gene expression for the two components of the transcription factor AP‐1, c‐fos and c‐jun. Despite the similar effects of TGF‐β1 on expression of cartilage matrix proteins and metalloproteases in this culture system, no appreciable changes in the expression of TGF‐β isoforms were evident in response to retinoic acid treatment. The present investigation demonstrates that regulation of AP‐1 responsive genes by retinoic acid can be either positive or negative, depending on the target cell type, and illuminates new mechanisms by which retinoic acid and other retinoids may exert control during development and growth of the limb. © 1994 wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Phorbol dibutyrate‐specific protein phosphorylation in brush border membranes of chicken enterocytes
Authors: Toskulkao Chaivat , Bhartur Sheela , Musch Mark W. , Chang Eugene B. , Rao Mrinalini C
Published: February 2005

We have demonstrated that phorbol esters such as phorbol dibutyrate (PhE) transiently inhibit Na/H exchange both in intact avian enterocytes and in brush border membrane (BBM) vesicles prepared from enterocytes treated with PhE (Chang et al., 1991, Am. J. Physiol. 260: C1264–C1272). Maximal inhibition occurs at 90 sec and values return to baseline by 15 mm. In this study we examined if PhE causes changes in BBM protein phosphorylation by two methods: (1) in situ phosphorylation in which intact cells prelabeled with 32Pi were treated with PhE; (2) in vitro phosphorylation in which BBM, isolated from untreated and PhE‐treated enterocytes, were exposed to γ32P‐ATP. In situ phosphorylation studies showed that, at 90 sec, PhE increases the phosphorylation of BBM proteins of Mr (pl): 150 (6.5), 89 (≈6.2), and 48 (≈6.1) kDa which declined to control values at 15 min, suggesting that these may be transport‐related substrates. These labeled substrates were recovered in the detergent‐insoluble fraction after extraction with 0.1% Triton X‐100 overnight. Transient phosphorylation of a number of proteins was also observed when BBM prepared from control or PhE‐treated cells were incubated with γ 32P‐ATP ± 10 nM PhE, phosphatidyl serine, Ca2+, and/or exogenous protein kinase C (PKC). The in vitro phosphoproteins included both Triton‐soluble and Triton‐insoluble proteins. However, none of these proteins labeled in vitro coincided with those labeled in situ. The decline in phosphorylation with time can be accounted for by phosphatase action as these BBM possess a Ca‐dependent phosphatase. In summary, we have demonstrated that the BBM possess PKC‐specific substrates which can be visualized by in situ and in vitro phosphorylation. Treatment of intact enterocytes with PhE results in the phosphorylation of three detergent‐insoluble proteins with a time course similar to that of PhE inhibition of Na/H transport. © 1994 wiley‐Liss, Inc.
Sulfation of Guinea pig megakaryocyte and platelet proteins
Authors: Schick Barbara P. , Jacoby Jennifer A.
Published: February 2005

This study has explored the sulfation of proteins by guinea pig megakaryocytes and platelets and by human platelets. Guinea pig megakaryocytes were incubated in vitro with [35S]sulfate, and the sulfated proteins were separated from proteoglycans by DEAE‐Sephacel chromatography and analyzed by SDS‐PAGE. The megakaryocytes esterified sulfate to a number of proteins, with the most extensive label migrating at Mr 42,000, and a second heavily labeled band at Mr 103,000 in the 0.1 M NaCl DEAE eluate, and 50 and 180 kDa in the 0.23 M NaCl eluate. [35S]‐Labeled GPIbα was immunoprecipitated from megakaryocyte Triton X‐100 extracts. Guinea pig platelet proteins were labeled in vivo by injection of the animals with a single dose of H235SO4. The platelets were activated with thrombin, and cytoskeletal proteins were isolated after treatment of the activated platelets with Triton X‐100. About 20% of the platelet macromolecule‐associated [35S]sulfate was incorporated into sulfated proteins, which were recovered primarily in the cytoskeleton. The cytoskeleton‐associated sulfate radiolabel migrated on SDS‐PAGE primarily with actin and additionally with several higher molecular weight proteins. A Mr 42,000 [35S]‐labeled protein was immunoprecipitated by a monoclonal anti‐actin antibody, along with molecules of Mr 160,000 and 180,000 and some higher Mr material, from the megakaryocytes labeled in vitro with [35S]sulfate. Actin was labeled on 2D isoelectric focusing/SDS‐PAGE gels. In addition, there was a very acidic series of heavily [35S]‐labeled 42 kDa proteins with about eight components of different isoelectric points with a pattern identical to the Mr 40,000 cytoskeletal‐associated glycoprotein Pltpg40 isolated by Hildreth et al. (1991, Blood 77:121). We hypothesize that sulfation of the cytoskeletal proteins might be involved in cytoskeletal protein interactions and function. © 1994 wiley‐Liss, Inc.
Binding of hyaluronan to plasma fibronectin increases the attachment of corneal epithelial cells to a fibronectin matrix
Authors: Nakamura Masatsugu , Mishima Hiroshi , Nishida Teruo , Otori Toshifumi
Published: February 2005

We wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01–10.0 μg/ml). Single‐cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1–1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell‐binding domain of fibronectin (FN12–8 or FN30–8, 0.03–0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin‐ and heparin‐binding domain at the N‐terminal (FN9–1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin‐ and heparin‐binding domain at the N‐terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N‐terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin‐ and heparin‐binding domain at the N‐terminal of plasma fibronectin and facilitates the attachment of epithelial cells. © 1994 wiley‐Liss, Inc.
Modulation of keratin 14 and α‐fetoprotein expression during hepatic oval cell proliferation and liver regeneration
Authors: Bisgaard Hanne Cathrine , Nagy Ton Peter Phuongnga T. , Hu Zongyi , Thorgeirsson Snorri S.
Published: February 2005

Keratin 14 (K14) expression has recently been demonstrated in cell lines of non‐parenchymal hepatic origin (Bisgaard et al., 1993, Mol. Carcinog., 7:60–66; Bisgaard et al., 1991, J. Cell. Physiol., 147:333–343). These cell lines are thought to represent a progeny of a dormant stem cell compartment present in the adult rat liver, which may participate in the restoration of the liver mass after experimental liver injury. Utilizing a combination of 2‐acetylaminofluorene (2‐AAF) administration and partial hepatectomy to activate liver regeneration by proliferation of oval cells, we examined the modulation of K14 as well as α‐fetoprotein (AFP) expression in proliferating oval cells and lineages hypothesized to be derived herefrom. We showed by Northern blot and in situ hybridization analyses that K14 and AFP transcripts were initially accumulating in epithelial cells located in subsets of ductal structures in the portal areas. As oval cells infiltrated the liver parenchyma, K14 transcripts were detected in oval cells, in foci of small basophilic hepatocytes, and in structures resembling glandular intestinal‐type epithelium. AFP was expressed in oval cells, and at low but detectable levels in foci of basophilic hepatocytes, but not in glandular intestinal‐type epithelium. Neither K14 nor AFP transcripts were detected in bile ducts or mature hepatocytes at any time during oval cell proliferation and reconstitution of the liver mass. To further study the modulation of K14 and AFP expression we utilized an in vitro model in which spontaneous transformation of rat liver epithelial (RLE) cells appeared to mimic the process of early differentiation along the hepatic lineage in vivo. We demonstrated that undifferentiated RLE cells at a late passage expressed K14 and vimentin, whereas transformation and differentiation to hepatoblast‐like progeny resulted in an abrogation of K14 and vimentin expression and an induction of K18 and AFP. We propose that K14 and AFP are sequentially modulated in subpopulations of oval cells involved in the ongoing reconstitution of the liver mass. © 1994 wiley‐Liss, Inc.
Expression of calmodulin and calmodulin binding proteins in lymphoblastoid cells
Authors: Colomer Josep , Agell Neus , Engel Pablo , Bachs Oriol
Published: February 2005

Calmodulin is encoded in vertebrates by three different genes: CALM1, CALM2, and CALM3. We have examined the mRNAs expressed from these three genes in eight lines of human lymphoblastoid cells (Namalwa, Raji, Ramos, JY, Molt‐4, Jurkat, CEM, and HPB‐ALL). We found that all these cell lines (except Ramos) overexpressed CALM3 transcripts, which led to an increase of total CaM protein with respect to quiescent normal I lymphocytes. The nuclear concentration of calmodulin was measured in two of these lymphoblastoid cell lines (JY and HPB‐ALL) and compared to quiescent and phytohemagglutinin‐activated T lymphocytes. Activated lymphocytes showed a 2‐fold increase of nuclear calmodulin with respect to quiescent cells, whereas in the two lymphoblastoid cell lines, nuclear calmodulin remained similar to that of quiescent cells. The levels of a calmodulin‐binding protein of 150 kDa in the homogenates of the eight lymphoblastoid lines was found to be higher than those of quiescent and activated lymphocytes. Likewise, the amount of three calmodulin‐binding proteins of 240, 200, and 170 kDa was also increased in several of the cell lines, but not in all of them. The 170‐kDa protein was only expressed by activated lymphocytes and lymphoblastoid cells, suggesting that it could be specific for proliferating cells. In the nuclei of activated lymphocytes and lymphoblastoid cells, a decrease of a calmodulin‐binding protein of 110 kDa and increases of three other of 240, 180 and 170 kDa were also detected. © 1994 wiley‐Liss, Inc.
Serum‐Induced vascular smooth muscle cell elastolytic activity through tyrosine kinase intracellular signalling
Authors: Kobayashi Jun , Wigle Dennis , Childs Tim , Zhu Li , Keeley Fred W. , Rabinovitch Marlene
Published: February 2005

In previous studies, we related increased elastolytic activity in pulmonary arteries (PA) with endothelial injury to the later development of PA hypertension in rats. As the mechanism causing the incrased PA elastase was unknown, we hypothesized that serum factors which are accessible to vascular smooth muscle cells (SMC) following endothelial injury stimulate their elastolytic activity. To test this, we developed an in vitro assay in which we added [3H]‐elastin to cultured vascular SMC after 24 h serum starvation and monitored elastolysis following a further 24 h incubation with fetal bovine serum (FBS). We observed that serum induced increased elastolytic activity in both PA and aorta‐derived SMC but not in endothelial cells or SMC with low basal levels of elastolytic activity. Maximum stimulation of SMC elastolytic activity occurred with a concentration as low as 1% FBS and despite elastase inhibitors in serum, suggesting that the activity is confined to the immediate pericellular region where enzyme concentration is high. Serum‐stimulated elastolytic activity was not reproduced by growth factors or cytokines known to be associated with vascular disease or to induce release of elastases in other cells. The serum inducing elastolytic activity was heat and acid labile. It was associated with increased elastin adhesion to the 67 kD elastin binding protein on SMC surfaces and was prevented by tyrosine kinase inhibitors but not protein kinase C or A inhibitors. Our studies therefore suggest a mechanism whereby serum induction of SMC elastase requires signalling through the elastin binding protein and activation of tyrosine kinase. © 1994 Wiley‐Liss, Inc.
Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing
Authors: Ning Shoucheng , Hahn George M.
Published: February 2005

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell–cell contact plays in this phenomenon. The formation of cell–cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca2+restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell–cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley‐Liss, Inc.
TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype
Authors: Breen Ellen C. , Ignotz Ronald A. , McCabe Laura , Stein Janet L. , Stein Gary S. , Lian Jane B.
Published: February 2005

This study examines the mechanism by which TGF‐β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre‐osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c‐fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF‐β1‐treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF‐β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF‐β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF‐β1 response throughout the course of osteoblast differentiation. The initiation of a TGF‐β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF‐β1. These in vitro results indicate that TGF‐β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley‐Liss, Inc.
Proinflammatory cytokines (interleukin‐1β and tumor necrosis factor‐α) down regulate synthesis and secretion of thrombospondin by human endothelial cells
Authors: Morandi Verônica , Cherradi Salah‐Eddine Lamhamedi , Lambert Sylvie , Fauvel‐Lafève Françloise , Legrand Yves J. , Legrand Chantal
Published: February 2005

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) by cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin‐ 1β (IL‐1β) or human tumor necrosis factor‐α (TNF‐α) caused a time‐ and dose‐dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL‐1β and TNF‐α in combination had a greater effect than either agent alone. Interferon‐γ (IFN‐γ) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with IL‐1β, either alone or in combination with TNF‐α, had no significant effect on the steady‐state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL‐1β and TNF‐α can selectively modulate the composition of the extracellular matrix by decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.
Transforming growth factor β1‐mediated induction of junB is selectively inhibited by expression of Ad.12‐E1A
Authors: Coussens Lisa M. , Chiu Robert , Yokoyama Kazushige
Published: February 2005

Transforming growth factor beta (TGF‐β), a multifunctional polypeptide growth factor, regulates the expression of many genes critical to cell cycle progression, such as members of the jun gene family which encode components of the transcription factor complex AP‐1. The transforming proteins encoded by the early region 1A of adenovirus12 (Ad. 12‐E1A) abrogate some of the cellular responses to TGF‐β as well as affecting, differentially, the expression of cellular jun genes. Our data demonstrate that expression of Ad. 12‐E1A in rat 3Y1 fibroblast cells inhibits induction of junB by TGF‐β1 while not altering the regulation of junB by phorbol ester or serum. Regulation of c‐jun gene expression by TGF‐β1, phorbol ester, and serum is not appreciably altered by the expression of Ad. 12‐E1A. Inhibition of TGF‐β induced junB expression is not due to a defect in TGF‐β/receptor interaction on Ad. 12‐E1A transformed cells and is not observed in other isotypic fibroblast cells transformed by SV40 or polyomavirus. These data suggest that multiple, independent, intracellular signal transduction pathways exist which mediate genomic responses to TGF‐β. Cellular expression of Ad. 12‐E1A‐12S gene products results in selective disruption of some TGF‐β1 signaling cascades and not those activated by phorbol ester or serum. These data further suggest that some cellular targets which mediate TGF‐β1 action may also be unique targets of action for the E1A‐12S transforming protein of adenovirus12. © 1994 Wiley‐Liss, Inc.
Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: Dependence on cell size, time from birth, insulin‐like growth factor I, and auto‐activation of protein Kinase C
Authors: Dempsey Edward C. , Badesch David B. , Dobyns Emily L. , Stenmark Kurt R.
Published: February 2005

Based on the unique susceptibility of the neonatal pulmonary circulation to hypoxia‐induced structural alteration in vivo, we hypothesized that pulmonary artery (PA) smooth muscle cells (SMC) from the neonate would demonstrate enhanced growth capacity in vitro compared to adult cells. To test this hypothesis, matched neonatal and adult bovine SMC were tested for differences in size, serum‐stimulated proliferation, susceptibility to senescence, resistance to serum withdrawal, autocrine growth capacity, and responsiveness to a locally important growth factor (insulin‐like growth factor I; IGF‐I) and an activator of protein kinase C (PKC) (phorbol 12‐myristate 13‐acetate; PMA). Neonatal PA SMC were smaller, grew faster, reached a higher plateau density, and were less susceptible to senescence. They were more resistant to serum withdrawal, had spontaneous autocrine growth capacity, and were more responsive to IGF‐I, PMA, and the combination. Acquisition of increased growth factor responsiveness occurred between d5 and d14 after birth. Increased neonatal growth to IGF‐I was associated with reduced IGF‐I binding activity, implicating a post‐receptor mechanism in enhanced responsiveness. Increased membrane‐bound PKC catalytic activity was found in serum‐deprived neonatal SMC. This basal increase was equal to that stimulated by 1 nM PMA in adult SMC, a pretreatment that caused these cells to become as responsive to IGF‐I as untreated neonatal ones. We conclude that neonatal bovine PA SMC have marked enhancement of growth capacity in vitro, the acquisition of which is dependent on time from birth and is associated with auto‐activation of PKC, These increased growth properties detected in vitro may contribute to the striking hyperplasia of neonatal PA SMC found in vivo following hypoxic exposure. © 1994 Wiley‐Liss, Inc.
Intracellular signal transduction for serum activation of the hyaluronan synthase in eukaryotic cell lines
Authors: Klewes Ludger , Prehm Peter
Published: February 2005

Hyaluronan synthase was activated in B6 cells or 3T3 fibroblasts by foetal calf serum with maximal activity after 6 h. Activation was inhibited by cycloheximide or by the protein kinase inhibitors H‐7 or H‐8, indicating that transcription as well as phosphorylation was required for activation. The activation by serum was markedly prolonged, when serum was added together with cholera toxin or theophylline. Without serum stimulation the hyaluronan synthase could also be activated by phorbol‐12‐myristate‐13‐acetate, by dibutyryl‐c‐AMP, or by forskolin. Increasing the intracellular Ca‐ion concentration with a Ca‐ionophore also led to an activation. The activation of the drugs was not synergistic. In isolated plasma membranes the synthase activity could be decreased by phosphatase treatment and enhanced by ATP in B6 cells and by ATP in the presence of phorbol‐12‐myristate‐13‐acetate in 3T3 fibroblasts. Stimulation correlated with increased transcription and phosphorylation of the 52 kD hyaluronan synthase at serine residues. The results led to the conclusion that hyaluronan synthase is induced by transcription and activated by phosphorylation by protein kinase C, c‐AMP‐dependent protein kinases, or Ca‐ion‐dependent protein kinases. © 1994 Wiley‐Liss, Inc.
Retinoids regulate expression of the retinol‐binding protein gene in hepatoma cells in culture
Authors: Mourey Marie Sophie , Quadro Loredana , Panariello Luigi , Colantuoni Vittorio
Published: February 2005

The expression of the gene coding for retinol‐binding protein has been studied in a system of cultured human hepatoma cells exposed to retinoids. We report that the gene is positively modulated by retinol and retinoic acid in a time‐ and dose‐dependent fashion. The stimulation at the mRNA level is paralleled by an increase of the corresponding protein that is secreted in the presence of the physiological ligand. An RBP‐CAT chimeric gene, introduced by transfection, is also responsive to the treatment, showing the gene dose‐dependency as the endogenous gene. These results demonstrate that retinoids up‐regulate the RBP gene and that the control takes place at transcriptional level. © 1994 Wiley‐Liss, Inc.
Tumor cells secrete an Angiogenic factor that stimulates basic fibroblast growth factor and Urokinase expression in Vascular Endothelial cells
Authors: Peverali Fiorenzo A. , Mandriota Stefano J. , Ciana Paolo , Marelli Rosaria , Quax Paul , Rifkin Daniel B. , Valle Giuliano Della , Mignatti Paolo
Published: February 2005

Culture medium conditioned by human SK‐Hep1 hepatoma cells or mouse S180 sarcoma cells rapidly up‐regulates endothelial cell expression of basic fibroblast growth factor (bFGF) and induces formation of a capillary‐like structures by vascular endothelial cells grown on three‐dimensional fibrin gels (in vitro angiogenesis). Incubation of endothelial cells with the tumor cell‐conditioned media also results in increased expression of urokinase plasminogen activator (uPA), a key component of the proteolytic system required for cell invasion and capillary formation. Although the tumor cell‐conditioned media contain no bFGF, addition of anti‐recombinant bFGF IgG abolishes the up‐regulation of uPA and blocks in vitro angiogenesis. This indicates that both the increase in uPA production and formation of capillary‐like structures are mediated by endogenous bFGF expressed by the endothelial cells. Both the bFGF/uPA‐inducing activity and the angiogenic activity of SK‐Hep1 cell‐conditioned medium copurify with a relatively acid‐resistant peptide that has moderate affinity for heparin and Mr < 18 kDa > 3.5 kDa. Known cytokines with similar biochemical features do not possess the same biological activity. These findings indicate that angiogenesis can be mediated by endothelial cell bFGF through an autocrine mechanism and that the bFGF‐inducing peptide may represent a novel tumor‐derived angiogenic factor that modulates in endothelial cells the concerted expression of cytokines and proteolytic enzymes required for capillary formation. © 1994 Wiley‐Liss, Inc.
Dog platelets accumulate intracellular Fibrinogen as they age
Authors: Heilmann E. , Hynes L. A. , Friese P. , George J. N. , Burstein S. A. , Dale G. L.
Published: February 2005

The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor‐mediated endocytosis or fluid‐phase pinocytosis. Despite many detailed studies on the function and content of α‐granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of α‐granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 ± 2% and 119 ± 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombosponding and P‐selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence‐activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 ± 23% of the level for the entire platelet population. These data demonstrate that age‐dependent changes in exogenously acquired α‐granule proteins do occur and that the uptake mechanism for these proteins is active through out the platelet life span. © 1994 Wiley‐Liss, Inc.
cAMP modulates stress protein synthesis in human Monocytes‐Macrophages
Authors: Pizurki Lara , Polla Barbara S.
Published: February 2005

The synthesis of heat‐shock proteins (HSPs) and other stress proteins, including heme oxygenase (HO) and ferritin, is differentially induced by heat and oxidizing agents. In order to determine what role cAMP plays in those inductions in human monocytes‐macrophages (mϕ), we used cAMP activators or analogues alone or in combination with various stressful conditions. A stimulation in cAMP production did not per se affect stress proteins synthesis in mϕ but modulated their induction in a differential way according to the stimulus. cAMP increased the synthesis of HSPs after heat shock. During erythrophagocytosis, whereas cAMP depressed the phagocytic process and the associated generation of superoxide anions, it enhanced the synthesis of HSPs, while inhibiting that of HO and ferritin. These results indicate that cAMP has a direct enhancing effect on the expression of stress proteins controlled by a classic heat‐shock promoter, while decreasing their expression when induced by oxidative stress. © 1994 Wiley‐Liss, Inc.
Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: Possible function in insulin processing in the blood–brain barrier
Authors: Miller Donald W. , Keller Bradley T. , Borchardt Ronald T.
Published: February 2005

The binding of 125 I‐insulin to primary cultures of bovine brain microvessel endothlial cells was examined. Insulin binding was both time and temperature dependent and inhibited by excess unlabeled insulin. Furthermore, the specific binding of insulin was polarized to the apical side of the cell monolayers. Upon binding, the labeled insulin was internalized, with approximately 70% resistant to acid wash over a 90‐min period. The inhibition of insulin internalization observed with cell monolayers exposed to either phenylarsine oxide or unlabeled insulin suggests a receptor‐mediated endocytic process. Furthermore, the ability of chloroquine to reduce the metabolism of insulin indicates a significant portion of the peptide iis processed through a lysosomal pathway. In contrast to the fluid‐phase endocytosis marker, Lucifer yellow, as much as 65% of internalized insulin undergoes apical to basolateral trancytosis in brain microvessel endothelial cells. While most of the effluxed insulin was degraded, as assessed by trichloroacetic acid precipitation, the results of the present study suggest insulin receptors within the brain microvasculature may be involved in the processing and transport of bloodborne insulin. © 1994 Wiley‐Liss, Inc.
Ornithine decarboxylase gene expression is aberrantly regulated via the cAMP signal transduction pathway in malignant H‐ras transformed cell lines
Authors: Hurta Robert A. R. , Wright Jim A.
Published: February 2005

We have tested the hypothesis that H‐ras transformed cells contain alterations in signal pathways important in controlling the expression of ornithine decarboxylase (ODC), the highly regulated rate‐limiting activity in the biosynthesis polyamines. Mouse 10 T 1/2 fibroblasts and a series of 10 T 1/2 H‐ras transformed cell lines were treated with stimulators of cAMP synthesis (forskolin and cholera toxin), a biologically stable analogue of cAMP (8‐bromo‐cAMP), and an inhibitor of cAMP degradation (3‐isobutyl‐1‐methylxanthine). Elevations in ODC gene expression were noted in H‐ras transformed cells that were not observed in parental 10 T 1/2 fibroblasts. The forskolin‐mediated effects were not detected with 1,9‐dideoxyforskolin, a compound structurally related to forskolin, which does not activate adenyl cyclase. The effects observed with cholera toxin were not detected when cells were treated with the purified subunits of this compound, indicating that the toxin‐induced effects were cAMP‐specific. Actinomycin D treatment prior to forskolin exposure reduced the elevation observed in ODC gene expression indicating the involvement of the transcriptional process. Furthermore, we observed that cycloheximide treatment of malignant but not benign H‐ras transformed cells significantly elevated ODC message level. Treatment of malignant cells with both cycloheximide and forskolin together resulted in a further additive elevation in ODC message, but a similar treatment of benign tumor cells reduced the forskolin‐mediated increase in ODC message. In addition, treatment of H‐ras transformed cell with the tumor promoter, 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10 T 1/2 fibroblasts. Cells treated with a combination of TPA and forskolin. Exposure of H‐ras transformed cells to TPA and forskolin together led to further pronounced increases in ODC message when compared to treatment with TPA or forskolin alone. These results demonstrate aberrant regulation of signal pathways involved in controlling ODC gene expression in H‐ras transformed cells and provide further insight into the altered growth regulatory program associated with malignant transformation. © 1994 Wiley‐Liss, Inc.
Glandular‐like morphogenesis and secretory activity of human tracheal gland cells in a three‐dimensional collagen gel matrix
Authors: Jacquot Jacky , Spilmont Christophe , Burlet Henriette , Fuchey Claudette , Buisson Anne Cecile , Tournier Jean Marie , Gaillard Dominique , Puchelle Edith
Published: February 2005

The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three‐dimensional (3‐D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland‐like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well‐developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland‐like tubules. A cell‐associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland‐like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3‐D collagen matrix form functional tracheal gland‐like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling. © 1994 Wiley‐Liss, Inc.