Growth Factor-stimulated Cell Proliferation Research Articles

Nuclear eNOS interacts with and S-nitrosates ADAR1 to modulate endothelial gene expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Collaborative Research Centre (CRC 1366); Chinese Scholarship Council (CSC) Introduction and Aim Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. The effects of NO are attributable to its interaction with heme-containing proteins e.g., soluble guanylyl cyclase, or to the S-nitrosation of proteins. Most eNOS is localized to the cell membrane or the Golgi apparatus, however, it has also been detected in the nucleus. In this study, we assessed the role of nuclear eNOS in endothelial cells. Methods and Results Confocal microscopy studies confirmed the presence of eNOS in the nucleus of unstimulated human and murine endothelial cells (ex vivo and in vitro) and stimulation with vascular endothelial growth factor (VEGF) increased eNOS nuclear translocation. Co-immunoprecipitation studies coupled with proteomics revealed the VEGF-dependent association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among these, nuclear eNOS bound double-stranded RNA-specific adenosine deaminase 1 (ADAR1), an enzyme involved in RNA editing via the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). ADAR1 was S-nitrosated in human endothelial cells and the knockdown of eNOS was associated with an increase in dsRNA (immunofluorescence) and altered ADAR1-mediated A-I editing (RNA seq). The accumulation of dsRNA in human endothelial cells lacking eNOS led to the activation of the interferon α/β signalling pathway (e.g., MX2, OASL, OAS1/2/3, RNASEL) and the downregulation of cell cycle-related genes. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as tumour necrosis factor- or H2O2-induced cell death were increased. Furthermore, the increased dsRNA in endothelial cells lacking eNOS elicited the activation of interferon-induced, dsRNA-activated protein kinase, which in turn phosphorylated the eukaryotic translation initiation factor 2A. Accordingly, new protein synthesis was inhibited following eNOS knockdown (immunofluorescence and mass spectrometry). Conclusion These results demonstrated that eNOS/NO signalling regulates endothelial type I interferon signalling and affects translation, cell cycle and cell death through S-nitrosation of ADAR1.

Glutathione and Glutaredoxin in Redox Regulation and Cell Signaling of the Lens.

The ocular lens has a very high content of the antioxidant glutathione (GSH) and the enzymes that can recycle its oxidized form, glutathione disulfide (GSSG), for further use. It can be synthesized in the lens and, in part, transported from the neighboring anterior aqueous humor and posterior vitreous body. GSH is known to protect the thiols of the structural lens crystallin proteins from oxidation by reactive oxygen species (ROS) so the lens can maintain its transparency for proper visual function. Age-related lens opacity or senile cataract is the major visual impairment in the general population, and its cause is closely associated with aging and a constant exposure to environmental oxidative stress, such as ultraviolet light and the metabolic end product, H2O2. The mechanism for senile cataractogenesis has been hypothesized as the results of oxidation-induced protein-thiol mixed disulfide formation, such as protein-S-S-glutathione and protein-S-S-cysteine mixed disulfides, which if not reduced in time, can change the protein conformation to allow cascading modifications of various kinds leading to protein–protein aggregation and insolubilization. The consequence of such changes in lens structural proteins is lens opacity. Besides GSH, the lens has several antioxidation defense enzymes that can repair oxidation damage. One of the specific redox regulating enzymes that has been recently identified is thioltransferase (glutaredoxin 1), which works in concert with GSH, to reduce the oxidative stress as well as to regulate thiol/disulfide redox balance by preventing protein-thiol mixed disulfide accumulation in the lens. This oxidation-resistant and inducible enzyme has multiple physiological functions. In addition to protecting structural proteins and metabolic enzymes, it is able to regulate the redox signaling of the cells during growth factor-stimulated cell proliferation and other cellular functions. This review article focuses on describing the redox regulating functions of GSH and the thioltransferase enzyme in the ocular lens.

Preparation and Characterization of Liposomal Everolimus by Thin-Film Hydration Technique

In 10% to 40% of the cases of coronary stent implantation, patients face in-stent restenosis due to an inflammatory response, which induces artery thickening. Everolimus, a drug that inhibits growth factor-stimulated cell proliferation of endothelial cells, represents a promising alternative to prevent in-stent restenosis. In this study, everolimus was encapsulated by a film hydration technique in liposomes by using phosphatidylcholine and cholesterol at different ratios. As the ratio of cholesterol increases, it modulates the rigidity of the structure which can affect the encapsulation efficiency of the drug due to steric hindrance. Moreover, various lipid : drug ratios were tested, and it was found that as the lipid : drug ratio increases, the encapsulation efficiency also increases. This behavior is observed because everolimus is a hydrophobic drug; therefore, if the lipidic region increases, more drug can be entrapped into the liposomes. In addition, stability of the encapsulated drug was tested for 4 weeks at 4°C. Our results demonstrate that it is possible to prepare liposomal everolimus by film hydration technique followed by extrusion with high entrapment efficiency as a viable drug delivery system.

Primary Cilia as Signaling Hubs in Health and Disease.

Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer.

Endogenous Selenoprotein P, a Liver-Derived Secretory Protein, Mediates Myocardial Ischemia/Reperfusion Injury in Mice.

Selenoprotein P (SeP), a liver-derived secretory protein, functions as a selenium supply protein in the body. SeP has been reported to be associated with insulin resistance in humans through serial analysis of gene expression. Recently, SeP has been found to inhibit vascular endothelial growth factor-stimulated cell proliferation in human umbilical vein endothelial cells, and impair angiogenesis in a mouse hind limb model. In this study, the role of SeP in ischemia/reperfusion (I/R) injury has been investigated. SeP knockout (KO) and littermate wild-type (WT) mice were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion. The myocardial infarct area/area at risk (IA/AAR), evaluated using Evans blue (EB) and 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in SeP KO mice than in WT mice. The number of terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei was significantly lower in SeP KO mice than in WT mice. In addition, caspase-3 activation was reduced in SeP KO mice compared to that in WT mice. Furthermore, phosphoinositide 3-kinase/Akt and Erk levels were examined for the reperfusion injury salvage kinase (RISK) pathway. Interestingly, SeP KO significantly increased the phosphorylation of IGF-1, Akt, and Erk compared to that in WT mice after I/R. Finally, I/R-induced myocardial IA/AAR was significantly increased in SeP KO mice overexpressing SeP in the liver compared to other SeP KO mice. These results, together, suggest that inhibition of SeP protects the heart from I/R injury through upregulation of the RISK pathway.

Sulfated glycosaminoglycan derivatives affect the bioactivity of angiogenic growth factors

Event Abstract Back to Event Sulfated glycosaminoglycan derivatives affect the bioactivity of angiogenic growth factors Linda Koehler1, Sandra Rother1, Stephanie Moeller2, Matthias Schnabelrauch2, Vera Hintze1 and Dieter Scharnweber1 1 TU Dresden, Max Bergmann Center of Biomaterials, Germany 2 INNOVENT e.V., Biomaterials Department, Germany Introduction: Sulfated glycosaminoglycans (GAGs) are promising candidates for functional bio­ma­terials since their sulfate groups modulate the binding of growth factors, thereby influencing their biological activity profile. The interaction of different GAGs with several growth factors has previously been reported. These stu­dies demonstrated a sulfation- and carbohydrate backbone-dependent binding of GAGs resulting in an influence on the interaction of these mediators with recep­tors and therefore their bioactivity[1],[2]. Angiogenic growth factors, like vascular endothelial (VEGF) and basic fibroblast growth factor (bFGF) play an important role during wound healing[3],[4]. Heparin is known to distinctly regulate VEGF and bFGF signaling by altering their interaction with the respective receptors[4],[5]. Hence we hypothesize that VEGF and bFGF interaction with different GAGs influences their bioactivity depending on their degree of sul­fa­tion (D.S.) and carbohydrate backbone. This might lead to important implications for the use of GAGs as part of biomaterials for controlling angiogenesis during wound healing in health com­pro­mised patients. Materials and Methods: The interaction of VEGF and bFGF with different sulfated hyaluronan (sHA) and chondroitin sulfate (CS) derivatives was determined via surface plasmon resonance (SPR). VEGF or bFGF were im­mobilized onto a sensor chip surface and dif­fe­rent concentrations of solute GAGs were injected to reveal their binding strength. Addi­tionally, ELISA experiments with immobilized GAGs were performed to validate the SPR results. The influence of GAGs on the capability of growth factors to bind to their receptors was again investigated by SPR, while the consequences of the GAG/growth factor interaction on bioactivity were studied in cell-based bioassays. Here pre-incubated GAG/growth factor complexes were added to the cells and growth factor-stimulated cell proliferation was de­termined. Results and Discussion: In case of bFGF the binding strength of sHA and CS derivatives reveals a strong dependence on the D.S. but not on the carbohydrate backbone of the GAG. For VEGF, however, low-sHA and high-sulfated CS showed a comparable bin­ding strength. These results imply that the different molecular geometries in the carbohydrate backbone of the GAGs might render the respec­tive sulfate groups to interact differently. This is further emphasized in investigations with TGF-β1 whe­re naturally sulfated heparin demonstrated a comparable binding strength to high sulfated CS, even though it has a lower D.S[2]. The binding of growth factors to their respective recep­tors was modulated in the presence of GAGs which implies an altered biological profile. This could be verified in cell-based bioassays, since the presence of GAGs affected the growth factor-stimulated proliferation of the cells. Conclusion: The results imply that sulfated GAGs are promising candidates to be included in biomaterial coatings for controlling angiogenesis and therefore wound hea­ling processes, which would in particular be beneficial for health com­pro­mised patients. We acknowledge financial support by the DFG [SFB Transregio 67, projects A2, A3 and Z3].

Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

Automatic generation of causal networks linking growth factor stimuli to functional cell state changes

Despite the increasing number of growth factor-related signalling networks, their lack of logical and causal connection to factual changes in cell states frequently impairs the functional interpretation of microarray data. We present a novel method enabling the automatic inference of causal multi-layer networks from such data, allowing the functional interpretation of growth factor stimulation experiments using pathway databases. Our environment of evaluation was hepatocyte growth factor-stimulated cell migration and proliferation in a keratinocyte-fibroblast co-culture. The network for this system was obtained by applying the steps: (a) automatic integration of the comprehensive set of all known cellular networks from the Pathway Interaction Database into a master structure; (b) retrieval of an active-network from the master structure, where the network edges that connect nodes with an absent mRNA level were excluded; and (c) reduction of the active-network complexity to a causal subnetwork from a set of seed nodes specific for the microarray experiment. The seed nodes comprised the receptors stimulated in the experiment, the consequently differentially expressed genes, and the expected cell states. The resulting network shows how well-known players, in the context of hepatocyte growth factor stimulation, are mechanistically linked in a pathway triggering functional cell state changes. Using BIOQUALI, we checked and validated the consistency of the network with respect to microarray data by computational simulation. The network has properties that can be classified into different functional layers because it not only shows signal processing down to the transcriptional level, but also the modulation of the network structure by the preceeding stimulation. The software for generating computable objects from the Pathway Interaction Database database, as well as the generated networks, are freely available at: http://www.tiga.uni-hd.de/supplements/inferringFromPID.html.

Abstract 1089: Two nonselective calcium channels, TRPC3 and TRPC6, play roles in erythropoietin signaling

Abstract The signal transduction pathways of erythropoietin (Epo), a glycoprotein regulating production of red blood cells, have been a model for understanding growth factor stimulated cell proliferation and differentiation. Erythropoietin receptors (Epo-R) are also expressed on non-hematopoietic cells, and recently Epo was reported to have antiapoptotic effects on these cells including a potential role in specific cancers. Two calcium channels were identified which are involved in Epo signaling, TRPC3 and TRPC6. These are homologous, nonselective calcium channels and are members of the canonical transient receptor potential (TRPC) subfamily expressed on human erythroblasts. Both channels have also been implicated in stimulating proliferation of cancer cells; TRPC3 in ovarian cancer, TRPC6 in gliomas, and both channels in breast cancer. Here TRPC3 and TRPC6 function was studied in transfected human embryonic kidney (HEK)-293T cells, and in the human leukemia UT-7/Epo cell line. Measurement of [Ca2+]i was performed on single cells with Digital Video Imaging. TRPC3 is activated by erythropoietin, whereas TRPC6 is not. In fact, TRPC6 can inhibit Epo-induced activation of TRPC3. Domains regulating channel activation were studied here. The TRP domain of TRPC3 is required for its activation, and substitution of five amino acids in the TRPC3 TRP domain with those of TRPC6 significantly reduced the Epo-stimulated increase in [Ca2+]i in TRPC3-expressing cells. Substitution of the same amino acids in the TRPC6 TRP with those of TRPC3 did not enhance the response of TRPC6-expressing cells to Epo stimulation. However, substitution of the distal TRPC6 C-terminus (C2) with that of TRPC3 in addition to substitution of TRPC6 TRP with TRPC3 TRP resulted in a TRPC6 channel with Epo responsiveness similar to TRPC3. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites S681, S708, or S764 with corresponding TRPC6 sequence did not affect TRPC3 Epo-responsiveness. TRPC3, but not TRPC6, and chimeras expressing the distal TRPC3 C terminus showed significantly increased membrane insertion following Epo stimulation. This was associated with substantial cytosketetal localization of TRPC3 and chimeras expressing TRPC3C2 but not TRPC6. These data demonstrate the important role of the TRP domain in specificity of channel responsiveness to agonist stimulation, and the requirement for other sites for optimal response to agonists, such as those found in the distal C-terminus of TRPC3. A few mutations in critical channel sites could have an important effect on proliferation and survival of cells expressing Epo-R. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2011-1089

The Regulation of NADPH Oxidase and Its Association with Cell Proliferation in Human Lens Epithelial Cells

NADPH oxidase (NOX)-generated reactive oxygen species (ROS) are essential for growth factor-stimulated cell proliferation. In this study, the regulatory role of p22phox, a membrane subunit of NOX, in NOX activity and platelet-derived growth factor (PDGF) mitogenic signaling were examined. Human lens epithelial B3 (HLE B3) cell lines with p22phox overexpressed (p22-OE) and p22phox knockdown (p22-KD) were used as models. Cells stimulated with PDGF were compared with nonstimulated control cells. The relative NOX activity and intracellular ROS generation were detected by lucigenin-based assay and DCFH fluorescence, respectively. Cell proliferation was measured by BrdU and fluorescent nucleic acid staining assays. p22phox, P-JNK, P-ERK1/2, P-Akt, P-p38, p47phox, and P-PDGF receptor in cell lysates were detected by Western blot analysis with the respective specific antibodies. p22-OE showed higher NOX activity, PDGF-stimulated ROS generation, cell proliferation, and activation of signaling cascades of ERK1/2, JNK, and Akt over the control (vector alone). In contrast, p22-KD displayed opposite results. In addition, PDGF stimulated p47phox and Rac1 translocations and induced binding between p22phox and the cytosolic subunits of p47phox, p67phox, and p40phox. Overexpression of p22phox increased p22phox-p47phox binding, enhanced, and prolonged the phosphorylation of PDGF receptor at Tyr857 with a corresponding inhibition of the activity of the oxidation-sensitive low molecular weight protein tyrosine phosphatase (LMW-PTP). However, p22phox knockdown weakened p22phox-p47phox binding and largely diminished the activation of PDGF receptor with no inhibition of LMW-PTP. PDGF mitogenic action in HLE B3 cells depends on p22phox to regulate NOX activity, which affects PDGF receptor function for cell proliferation.

Mechanisms by Which S-Albuterol Induces Human Bronchial Smooth Muscle Cell Proliferation

Background: Racemic albuterol is a 50:50 mixture of the R-isomer, levalbuterol, and the S-isomer, S-albuterol. S-Albuterol increases airway hyperresponsiveness to spasmogens, exacerbates asthmatic conditions and stimulates cell growth, whereas levalbuterol attenuates cell growth in culture. The mechanisms of S-albuterol-induced cell proliferation are not well understood. We studied the role of albuterol isomers and intracellular cell cycle regulators on proliferation of human bronchial smooth muscle cells. Methods: Serum-starved cells (72 h) were fed test agents for 24 h and cell proliferation was measured. The expression of nuclear factor-ĸB inhibitory protein IĸBα, nuclear factor-ĸB, cyclin-dependent kinases 2 and 4, interleukin (IL)-6, and retinoblastoma and platelet-activating factor (PAF) receptor protein were measured by Western blotting. Results:S-Albuterol, PAF and platelet-derived growth factor stimulated cell proliferation, but levalbuterol and the racemic mixture inhibited cell proliferation compared with the effect of 5% fetal bovine serum alone. The proliferative effect of platelet-derived growth factor on S-albuterol was not additive, suggesting that the 2 mediators act by different mechanisms. S-Albuterol induced greater expression of all the measured proteins than either levalbuterol, the racemic mixture or 5% fetal bovine serum. S-Albuterol stimulated IL-6 secretion and abolished the ability of levalbuterol to inhibit IL-6 secretion. Conclusion: Our data show that S-albuterol stimulates cell proliferation by activating expression and phosphorylation of several intracellular mitogenic proteins and may exacerbate asthma by stimulating the release of IL-6. Induction of PAF receptor protein expression by S-albuterol strongly suggests that S-albuterol may exert its adverse effects by binding to a G protein-coupled receptor such as the PAF receptor.

Shp2E76K Mutant Confers Cytokine-independent Survival of TF-1 Myeloid Cells by Up-regulating Bcl-XL

Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gain-of-function Shp2 mutants such as Shp2E76K are associated with myeloid leukemias. We found that Shp2E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2E76K-induced cell survival mechanism in this study. Expression of Shp2E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-XL and Mcl-1 were up-regulated in Shp2E76K-transformed TF-1 (TF-1/Shp2E76K) cells. Knockdown of Bcl-XL but not Mcl-1 with short hairpin RNAs prevented Shp2E76K-induced cytokine-independent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2E76K cells, whereas the Bcl-XL inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2E76K-induced Bcl-XL expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2E76K cells. These results show that Shp2E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-XL through the Erk1/2 pathway.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Recent advances in Ca 2+-dependent Ras regulation and cell proliferation

Our understanding of the mechanisms whereby growth factors stimulate cell proliferation through the Ras pathway stems largely from studies of the canonical pathway involving recruitment of Ras activators and inhibitors to the vicinity of receptor tyrosine kinases via phosphotyrosine-binding adaptor proteins. Ca 2+ has seldom joined the party, despite the identification of phospholipase Cγ and Ca 2+ entry as receptor tyrosine kinase-dependent signals. Mechanisms by which Ca 2+ can directly influence Ras activity have remained relatively elusive. Similarly, the mechanisms whereby Ca 2+ modulates the cell cycle have been equally murky, and yet there are some interesting parallels in the role of Ras and Ca 2+ in cell cycle re-entry. This review focuses on a number of novel mechanisms that link Ca 2+ with the regulation of Ras activity and signaling output. Their collective discovery adds to the complexities of Ras regulation and raises further questions about the role of Ca 2+ signals in Ras-dependent cell proliferation.

Increased abundance of labile intracellular zinc during cell proliferation was due to increased retention of extracellular zinc in 3T3 cells

Platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and insulin-like growth factor I (IGF-I)-stimulated cell proliferation in 3T3 cells was accompanied by increased abundance of labile intracellular pool of zinc (LIPZ). However, the origin and regulation of this cell proliferation-associated increase in the abundance of LIPZ are unknown. Cellular zinc homeostasis involves zinc transporters and metallothionein. The objectives of this study were to determine whether cell proliferation-associated increase in the abundance of LIPZ was a result of an increased zinc uptake and to assess the involvement of zinc transporters and metallothionein in this cell proliferation-associated increase in the abundance of LIPZ in 3T3 fibroblasts. Zinc transporters assessed included both zinc importer (Zip1) and zinc exporters (ZnT1, ZnT2 and ZnT4). Growth factors increased the abundance of LIPZ while total cellular zinc concentration remained unaffected, demonstrating that LIPZ was responsive to the increased needs for zinc during growth factor-stimulated cell proliferation. Growth factors also increased net zinc retention as indicated by higher 65zinc radioactivity and elevated mRNA levels of Zip1, ZnT1 and ZnT4. Although zinc is essential to cell proliferation, excessive cellular zinc accumulation causes cytotoxicity. Collectively, these observations suggest that increase in the abundance of LIPZ during growth factor-stimulated cell proliferation was due to increased net retention of extracellular zinc, which was apparently achieved through a coordinated up-regulation of the expression of transporters involved in both zinc influx and efflux to ensure adequate supply of zinc to sustain cell proliferation, yet to prevent potential zinc cytotoxicity in 3T3 cells.

Heparin-binding epidermal growth factor-like growth factor stimulates cell proliferation in cerebral cortical cultures through phosphatidylinositol 3′-kinase and mitogen-activated protein kinase

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.

Insulin-activated, K+-channel-sensitive Akt pathway is primary mediator of ML-1 cell proliferation

Voltage-gated K(+) channel activities are involved in regulating growth factor-stimulated cell proliferation in a variety of cell types. Here we report that suppression of a voltage-gated K(+) channel with 4-aminopyridine (4-AP), barium, and tetraethylammonium inhibited both EGF- and insulin-stimulated myeloblastic leukemia ML-1 cell proliferation in a concentration-dependent manner. Both MAPK/ERK and Akt pathways are known to mediate cell proliferative signals of a variety of growth factors including insulin. In serum-starved ML-1 cells, insulin rapidly stimulated phosphorylation of ERK1/2 and Akt, and the phosphorylation levels peaked approximately 30 min after treatment. Pretreatment of ML-1 cells with 4-AP potently and dose-dependently prevented phosphorylation of ERK1/2 and Akt. However, insulin-induced activation of the Akt pathway also played a role in promoting ML-1 cell proliferation. Flow cytometry analysis revealed that although ML-1 cells were primarily arrested at G(1) phase by serum starvation for 36 h, they reentered the cell cycle after treatment with serum or insulin for 24 h. However, concomitant 4-AP treatment was able to attenuate cell cycle progression in synchronized ML-1 cells stimulated with growth factors. Our results strongly suggest that a 4-AP-sensitive K(+) channel activity plays an important role in controlling proliferation of ML-1 cells by affecting the activation of multiple signal transduction processes induced by insulin.

Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in cultured rat hepatic stellate cells: Yoshimasa Kobayashi, Shigeki Koide, Hirotoshi Nakamura, Hamamatsu University School of Medicine, Hamamatsu Japan

Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in cultured rat hepatic stellate cells: Yoshimasa Kobayashi, Shigeki Koide, Hirotoshi Nakamura, Hamamatsu University School of Medicine, Hamamatsu Japan

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells.

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine.

We describe a protein family in Drosophila containing six adenosine deaminase-related growth factors (ADGFs), which are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine.