bluuebunny/crossref_metadata_2025_split huggingface dataset:
Threonine 308 phosphorylated form of akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin
Authors: Borgatti Paola , Martelli Alberto M. , Tabellini Giovanna , Bellacosa Alfonso , Capitani Silvano , Neri Luca M. 
Published: May 2003

AbstractWe have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3‐kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co‐immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A. © 2003 Wiley‐Liss, Inc.
Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G‐protein‐coupled protease‐activated receptor‐1
Authors: Faraut Brice , Barbier Julien , Ravel‐Chapuis Aymeric , Doyennette Marie‐Agnès , Jandrot‐Perrus Martine , Verdière‐Sahuqué Martine , Schaeffer Laurent , Koenig Jeanine , Hantaï Daniel 
Published: May 2003

AbstractRegulation of thrombin activity may be required during skeletal muscle differentiation since the thrombin tissue inhibitor protease nexin‐1 appears at the myotube stage before being localized at the neuromuscular synapse. Here, we have used a model of rat fetal myotube primary cultures to study the effect of thrombin on acetylcholine receptor (AChR) expression, which is enhanced at the myotube stage. Our results show that thrombin decreases both the number of surface AChRs (AChRn) and AChR α‐subunit gene expression. Using the agonist peptide SFLLRN, we establish that the AChRn decrease is mediated by the G protein‐coupled thrombin receptor “protease‐activated receptor‐1” (PAR‐1). Moreover, the specific thrombin inhibitor hirudin increases AChRn by inhibiting the thrombin intrinsically present in the cultures. We further demonstrate that the activation of PAR‐1 by thrombin induces intracellular calcium movements that are blocked by 2‐APB, an inhibitor of inositol 1,4,5‐triphosphate (IP3)‐induced calcium release. These calcium signals are more intense in nuclei than in the cytoplasm and are consistent with the intracellular distribution of IP3 receptor that we find in the cytoplasm in a cross‐striated pattern and at a high level in the nuclear envelope zone. Finally, we show that the blockade of these IP3‐induced calcium signals by 2‐APB prevents the AChRn decrease induced by thrombin. Our results thus demonstrate that thrombin downregulates AChR expression by activating PAR‐1 and that this effect is mediated via an IP3 signaling pathway. © 2003 Wiley‐Liss, Inc.
Primary culture model of peroxisome proliferator‐activated receptor γ activity in prostate cancer cells
Authors: Xu Yue , Iyengar Sunita , Roberts Richard L. , Shappell Scott B. , Peehl Donna M. 
Published: May 2003

AbstractBRL 49653 (rosiglitazone) is a thiazolidinedione anti‐diabetic drug that activates the nuclear receptor, peroxisome proliferator‐activated receptor gamma (PPARγ). Pilot clinical trials have shown evidence of therapeutic activity of PPARγ agonists against prostate cancer. To more effectively use PPARγ ligands to treat this common and generally chemo‐resistant type of cancer, it will be necessary to better understand the nature of PPARγ activity in prostate cancer cells. Tumor suppressor effects of activation of PPARγ may include suppression of growth and/or induction of differentiation or apoptosis. We investigated responses of primary cultures of human prostatic cancer cells to BRL 49653. PPARγ was expressed in all of the cell strains examined. BRL 49653 caused dose‐ and time‐dependent growth inhibition that was associated with increased expression of the transcription repressor, transforming growth factor β‐stimulated clone 22 (TSC‐22), and markedly increased expression of the secretory differentiation‐associated gene adipophilin. Adipocyte‐type fatty acid binding protein (aFABP), neutrophil gelatinase‐associated lipocalin (NGAL), glycerol kinase (GyK), and β‐catenin, which are regulated by PPARγ ligands in certain other types of cells, were not regulated by BRL 49653 in prostate cells. Upregulation of adipophilin coincided with morphological changes and the appearance of cytoplasmic vacuoles with ultrastructural features of secondary lysosomes. These results extend previous studies with established cancer cell lines and show that PPARγ agonists can inhibit proliferation and modulate expression of secretory‐associated genes in primary cultures of prostate cancer cells, further warranting consideration of these agents as pro‐differentiating chemotherapeutic or chemoprevention agents for the treatment of prostate cancer. © 2003 Wiley‐Liss, Inc.
EGF receptor‐mediated, c‐Src‐dependent, activation of Stat5b is downregulated in mitogenically responsive hepatocytes
Authors: Guren Tormod K. , Ødegård John , Abrahamsen Hilde , Thoresen G. Hege , Susa Mira , Andersson Yvonne , Østby Eva , Christoffersen Thoralf 
Published: May 2003

AbstractSignal transducer and activator of transcription (STAT) proteins may be activated by epidermal growth factor (EGF), but their role in EGF receptor‐mediated mitogenic signaling is not clear. We previously showed that Stat5b was activated by EGF in rat hepatocytes in primary monolayer culture. In the present study, we found that EGF induced tyrosine phosphorylation of Stat5b both on Tyr‐699, which correlated with specific DNA binding activity, and also on other tyrosine residues. The Src tyrosine kinase inhibitor CGP77675 blocked the EGF‐induced activation of Stat5b, but did not affect the Stat5b activation by growth hormone (GH) or prolactin (PRL). The Stat5b response to EGF was most pronounced soon (3 h) after plating (early G1) and at high cell density (50,000 hepatocytes per cm2). However, at this cell density EGF did not stimulate DNA synthesis. In hepatocytes at 24 h of culturing (mid/late G1) with 20,000 cells per cm2, EGF induced strong phosphorylation of the EGF receptor, as well as Shc and ERK, and stimulated DNA synthesis, but did not activate Stat5b, although the Stat5b response to GH or PRL was retained. A strong GH‐induced Stat5b activation neither influenced the DNA synthesis alone nor enhanced the mitogenic effect of EGF. The results show that EGF induces tyrosine phosphorylation and DNA‐binding activity of Stat5b in a manner different from GH and PRL, apparently by a Src‐dependent mechanism. The data also provide further evidence that Stat5b is not required for mitogenic signaling from the EGF receptor. © 2003 Wiley‐Liss, Inc.
Post‐transcriptional expression of DMT1 in the heart of rat
Authors: Ke Ya , Chen Yin Yin , Chang Yan Zhong , Duan Xiang Lin , Ho Kwok Ping , Jiang De He , Wang Kui , Qian Zhong Ming 
Published: May 2003

AbstractNon‐transferrin‐bound iron (NTBI) overtaken by heart cells might be a key cause leading to iron‐mediated injury in heart disorders. NTBI uptake by heart cells might be mediated by divalent metal transporter 1 (DMT1). The understanding of the role of DMT1 in heart iron metabolism is fundamental for elucidating the cause resulting in excessive iron in the heart. The study was to evaluate effects of age and dietary iron on DMT1 mRNA expression and protein synthesis in rat heart. DMT1 mRNA expression was determined by RT‐PCR and sequence analysis, and DMT1 protein by Western blot analysis. DMT1 mRNAs with or without iron‐responsive element (IRE) both were found in rat heart. Expression of two forms of DMT1 mRNAs was the lowest at the age of post‐natal day (PND) 7, and then increased with the age, reaching the highest at PND196 (non‐IRE form) and PND63 (IRE form), respectively. During different ages, the levels of DMT1 (IRE) mRNA were higher than those of DMT1 (non‐IRE) mRNA and were significantly correlated with the non‐heme iron contents in the heart. After fed a high iron for 6 weeks, the rats had a sixfold elevation in heart iron and 22% (non‐IRE from) and 40% (IRE from) reduction in DMT1 protein compared to the controls. A low iron diet for 6‐weeks caused cardiac hypertrophy and heart iron deficiency and also an increase in levels of two forms of DMT1 proteins. However, iron status had no significant effect on DMT1 (IRE) and DMT1 (non‐IRE) mRNAs expression in the heart, although it can significantly influence heart transferrin receptor (TfR) mRNA expression. The results demonstrated that DMT1 mRNAs expression in the heart is age‐dependent and that two forms of DMT1 mRNAs both are regulated by iron on the post‐transcriptional level only. © 2003 Wiley‐Liss, Inc.
Induction of leukotriene C4 synthase after the differentiation of rat basophilic leukemia cells with retinoic acid and a low dose of actinomycin D and its suppression with methylprednisolone
Authors: Abe Masayoshi , Shibata Kazuhiko , Urata Hidenori , Sakata Noriyuki , Katsuragi Takeshi 
Published: May 2003

AbstractLeukotriene C4 synthase (LTC4 S) is a pivotal enzyme for generation of cysteinyl‐leukotrienes (cysLTs). LTC4 S activity in rat basophilic leukemia‐1 (RBL‐1) cells increased after culture in the presence of retinoic acid (RA) analogues, which was inhibited by cycloheximide or actinomycin D (ACD). Unexpectedly, the co‐addition of a low dose of ACD with RA further potentiated the upregulation of the LTC4 S activity. Daunorubicin and mitomycin C also had a similar effect. When stimulated with calcium ionophore A23187, control cells did not produce cysLTs, but RA‐treated cells generated cysLTs and the co‐addition of ACD further increased. While LTC4 S mRNA and protein increased in the cells treated with RA, the co‐addition of ACD further potentiated both in proportion to the LTC4 S activity. The effect of ACD was considered to enhance the transcription rate of LTC4 S gene, but not the mRNA‐stability. The addition of methylprednisolone (MP) inhibited generation of cysLTs from the cells with A23187‐stimulation and also did LTC4 S activity, but did not inhibit 5‐lipoxygenase (5‐LOX). The suppression of LTC4 S with MP showed a dependent manner on the time‐point and duration of MP‐treatment after RA‐addition which was correlated with reduction in LTC4 S mRNA and protein. The cells cultured with RA plus ACD contained more histamine, chymase activity, and granules in the cytoplasm than the control cells, suggesting differentiation to mature mast cells. In consideration of RA‐differentiation therapy, it may be of pathophysiological relevance that the antineoplastic agents potentiate RA‐induced, steroid‐sensitive, induction of LTC4 S in RBL‐1 cells. © 2003 Wiley‐Liss, Inc.
Functional activity of serotoninergic and melatoninergic systems expressed in the skin
Authors: Slominski Andrzej , Pisarchik Alexander , Zbytek Blazej , Tobin Desmond J. , Kauser Söbia , Wortsman Jacobo 
Published: May 2003

AbstractWe tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (5HT), melatonin (MT), and melatonin‐related receptors (MRR) showed expression of the isoforms 5HT2B, 5HT7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; 5HT2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and 5HT2B and documented the process of RNA editing for 5HT2C. Testing for functional activity of these receptors with serotonin and melatonin (10−14 to 10−10 M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum‐free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N‐acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10−10 M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability. © 2003 Wiley‐Liss, Inc.
Modulation of human fibroblast gap junction intercellular communication by hyaluronan
Authors: Moyer K.E. , Ehrlich H.P. 
Published: May 2003

AbstractThe composition of the extracellular matrix changes during dermal repair. Initially, hyaluronan (HA) concentration is high, however, by day 3, HA is eliminated. HA optimizes collagen organization within granulation tissue. One possible mechanism of HA modulation of collagen packing is through the promotion of gap junction intercellular communication (GJIC). Gap junctions are gated channels that allow rapid intercellular communication and synchronization of coupled cell activities. The gap junction channel is composed of connexin (Cx) proteins that form a gated channel between coupled cells. HA is reported to enhance Cx43 expression in transformed fibroblasts. GJIC was quantified by the scrape loading technique and reported as a coupling index. The coupling index for human dermal fibroblasts was 4.6 ± 0.2, while the coupling index for fibroblasts treated with HA more than doubled to 10.6 ± 0.7. By Western blot analysis no differences were appreciated in the protein levels of Cx43 or β‐catenin, a protein involved in the translocation of Cx to the cell surface. By immuno‐histology Cx43 and β‐catenin were evenly distributed throughout the cell in controls, but in cells treated with HA these proteins were co‐localized to the cell surface. Coupled fibroblasts are reported to enhance the organization of collagen fibrils. It is proposed that HA increases the accumulation of Cx43 and β‐catenin on the cell surface, leading to greater GJIC and enhanced collagen organization. © 2003 Wiley‐Liss, Inc.
Modulation of metabolic control by angiotensin converting enzyme (ACE) inhibition
Authors: Henriksen Erik J. , Jacob Stephan 
Published: May 2003

AbstractAngiotensin converting enzyme (ACE) inhibitors are a widely used intervention for blood pressure control, and are particularly beneficial in hypertensive type 2 diabetic subjects with insulin resistance. The hemodynamic effects of ACE inhibitors are associated with enhanced levels of the vasodilator bradykinin and decreased production of the vasoconstrictor and growth factor angiotensin II (ATII). In insulin‐resistant conditions, ACE inhibitors can also enhance whole‐body glucose disposal and glucose transport activity in skeletal muscle. This review will focus on the metabolic consequences of ACE inhibition in insulin resistance. At the cellular level, ACE inhibitors acutely enhance glucose uptake in insulin‐resistant skeletal muscle via two mechanisms. One mechanism involves the action of bradykinin, acting through bradykinin B2 receptors, to increase nitric oxide (NO) production and ultimately enhance glucose transport. A second mechanism involves diminution of the inhibitory effects of ATII, acting through AT1 receptors, on the skeletal muscle glucose transport system. The acute actions of ACE inhibitors on skeletal muscle glucose transport are associated with upregulation of insulin signaling, including enhanced IRS‐1 tyrosine phosphorylation and phosphatidylinositol‐3‐kinase activity, and ultimately with increased cell‐surface GLUT‐4 glucose transporter protein. Chronic administration of ACE inhibitors or AT1 antagonists to insulin‐resistant rodents can increase protein expression of GLUT‐4 in skeletal muscle and myocardium. These data support the concept that ACE inhibitors can beneficially modulate glucose control in insulin‐resistant states, possibly through a NO‐dependent effect of bradykinin and/or antagonism of ATII action on skeletal muscle. © 2003 Wiley‐Liss, Inc.
1–40 β‐amyloid protein fragment modulates the expression of CD44 and CD71 on the astrocytoma cell line in the presence of IL1β and TNFα
Authors: Speciale Livianna , Ruzzante Stefania , Calabrese Elena , Saresella Marina , Taramelli Donatella , Mariani Claudio , Bava Laura , Longhi Renato , Ferrante Pasquale 
Published: May 2003

AbstractThe modulation of CD44, VCAM‐1 and CD71 expression was analysed by flow cytometry in the 1321N1 astrocytoma cell line in the presence of interleukin‐1β (IL1β), tumour necrosis factor‐α (TNFα) and 1–40 or 25–35 β‐amyloid (Aβ) fragments. The percentage of 1321N1 astrocytoma cell line expressing these markers increased significantly after treatment with TNFα or IL1β. The presence of Aβ 1–40 fragment, alone or in combination with IL1β, induced an increase in the percentage of cells expressing CD44, but not VCAM‐1. However, the concomitant presence of Aβ 1–40 fragment and of IL1β or TNFα caused an increase in the percentage of CD71 positive cells. In contrast, the shorter Aβ 25–35 fragment was always inactive. These results indicates that Aβ 1–40 fragment, in association with cytokines, can activate this astrocyte‐derived cell line and add further elements in favour of the hypothesis that β‐amyloid can act as immunological mediator. © 2003 Wiley‐Liss, Inc.
Cdc42 and RhoA are differentially regulated during arachidonate‐mediated HeLa cell adhesion
Authors: Roberts Louis A. , Glenn Honor , Hahn Chang S. , Jacobson Bruce S. 
Published: May 2003

AbstractCell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5‐lipoxygenase to leukotrienes and cyclooxygenase‐1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen. © 2003 Wiley‐Liss, Inc.
Expression of connective tissue growth factor in bone: Its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo
Authors: Safadi Fayez F. , Xu Jie , Smock Steven L. , Kanaan Reem A. , Selim Abdul‐Hafez , Odgren Paul R. , Marks Sandy C. , Owen Thomas A. , Popoff Steven N. 
Published: May 2003

AbstractConnective tissue growth factor (CTGF) is a secreted, extracellular matrix‐associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti‐CTGF neutralizing antibody caused a dose‐dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo. © 2003 Wiley‐Liss, Inc.