Growth Factor-mediated Signal Transduction Research Articles

Role of Tyrosine Kinase Receptors in Growth Factor Mediated Signal Transduction, with Specific Reference to MAPK/Rasand p13k-Akt Containing Pathways in Oncogenesis: A Qualitative Database Review

Receptor Tyrosine kinases (RTKs) play a crucial role in the signal transduction pathways at cellular levels. RTK plays a vital role in cellular communication and transmission of signals to the adjacent cells and regulates different functions of the cell, such as cellular growth, differentiation, metabolism and motility. RTK s triggers growth factor receptors such as epidermal growth factor, insulin growth factor-1 receptor, platelet derived growth factor receptor, and fibro blast growth factor receptor and vascular endothelial growth factor receptor, thereby initiating and regulating cell growth and proliferation. MAPK/RAS and PI3/AKT pathways are the major pathways of RTK’s function. Dysregulation of these RTK’s and pathways often leads to many diseases such as Noonan Syndrome, Logius Syndrome, CFC syndrome and different types of cancer. Point mutation and over expression of receptors and mutations in Ras leads to 30% of human cancers. Also over expression of different growth factor receptors by RTK too lead to several types of cancers as Glioblastoma, Thyroid cancer, Colon cancer and Non-small cell lung cancer. PTEN mutation in PI3/AKT pathway often leads to carcinoma relative to Thyroid, Skin, Large intestine, eye and Bone. Therefore, these RTK’s often used as targets for cancer therapies. The medical sector uses various types of small molecule tyrosine kinase inhibitors such as ATP competitive inhibitors, Allosteric inhibitors and covalent inhibitors which are known as Afatinib, Crizotinib, Eroltinib, Icotinib, Lepatinib and Lenvatinib in treatment and management of differential carcinomas.

Brain-derived neurotrophic factor induces thioredoxin-1 expression through TrkB/Akt/CREB pathway in SH-SY5Y cells

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophic factors that are vital to the survival and proliferation of neuron. Thioredoxin-1 (Trx-1) is a redox regulating protein and plays various roles in regulating transcript factors and inhibiting apoptosis. It has been reported that Trx-1 is required for nerve growth factor-mediated signal transduction and neurite outgrowth, and is involved in synaptic protein expression induced by BDNF. However, the molecular mechanism on BDNF inducing Trx-1 expression has not been fully verified. The present study investigated the expression of Trx-1 after treatment with BDNF in SH-SY5Y cells. We first demonstrated that cell viability and the expression of Trx-1 were increased by BDNF in SH-SY5Y cells, which were inhibited by the tyrosine kinase B (TrkB) inhibitor, K252a, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002. Moreover, BDNF increased the activity of cAMP response element-binding protein (CREB) through TrkB/PI3-K/Akt pathway. Whereas the expression of Trx-1 induced by BDNF was suppressed by CREB siRNA. Thus, our data suggest that BDNF induces the expression of Trx-1 through the TrkB/Akt/CREB pathway.

Temporal control of growth factor‐mediated signaling pathways during cell differentiation and Xenopus embryonic development

Growth factor‐mediated signaling pathways regulate a wide‐spectrum of cellular functions such as survival, proliferation, differentiation, and apoptosis. Different growth factors typically trigger the activation of the same set of downstream signaling pathways which includes the mitogen activated protein kinase (MAPK), phosphoinositide 3‐kinase (PI3K)‐AKT, and phospholipase C (PLC) pathways Evidence suggests that precise spatiotemporal regulation of these signaling pathways is involved in generating diverse cellular outcomes. Although conventional genetic and pharmacological approaches have helped us understand different aspects of signaling pathways, a quantitative delineation of their signaling kinetics is pending due to a lack of tools that allow for precise temporal control of these pathways. Non‐neuronal optogenetics, an emerging technique that utilizes light to control intracellular signaling pathways, is a remarkable tool to help achieve this goal. Here, we introduce a bicistronic optogenetic system which we recently developed in our laboratory to achieve temporal control of the MAPK pathway. This tool uses the photoactivatable protein cryptochrome 2 (CRY2) and the N‐terminal domain of cryptochrome‐interacting basic‐helix‐loop‐helix (CIBN) from Arabidopsis thaliana. This novel system allows reversible, light‐mediated activation of the MAPK signaling pathway in intact mammalian cells as well as in developing Xenopus laevis embryos. In PC12 neuronal cells, light‐controlled, intermittent MAPK activity reveals a memory effect in light‐induced neurite outgrowth. In Xenopus embryos, developmental stage‐specific MAPK activation further reveals that this pathway can reprogram cell fate after a crucial time window when germ layers are specified. Our strategy can be generalized to control other kinase pathways with a similar activation mechanism. Results from our research will help resolve intracellular mechanisms of growth factor‐mediated signal transduction during cell differentiation and embryonic development.Support or Funding InformationKai Zhang, Department of Biochemistry, University of Illinois Urbana‐ChampaignThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Light-Controlled Growth Factor-Mediated Signal Transduction

Growth factor-mediated signal transduction regulates critical cellular responses including cell proliferation, differentiation, migration, and apoptosis. However, direct and quantitative linkage between time kinetics of growth factor-mediated pathways and the cellular response is still lacking due to difficulties in perturbing the kinetics of intracellular signaling pathways. Here, we construct an optogenetic system that uses light to regulate growth-factor mediated signaling pathways. We show that various downstream pathways control cell fate determination differentially. Overall, light-controlled growth-factor signal transduction enables precise dissection of individual subsets of signaling pathways in cells.

Abstract 4308: A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction.

Abstract Background: Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome and the fusion gene BCR-ABL, which has been shown to activate key signaling pathways, including PI-3 kinase, RAS/ERK and STAT5. ABL tyrosine kinase inhibitors (TKIs) are highly effective clinical agents that inhibit downstream signaling, trigger CML cell apoptosis, and provide clinical evidence for the phenomenon of “oncogene addiction," whereby cell survival becomes critically dependent upon the activity of a particular oncogene product. TKI therapy has largely failed to achieve the same degree of clinical success in other activated kinase-driven malignancies, suggesting that BCR-ABL is relatively unique in its ability to establish a state of oncogene addiction. Results: To dissect the mechanism by which BCR-ABL coordinates STAT5 and RAS/ERK signaling to establish a state of oncogene addiction, JAK2 kinase activity and RAS-GTP levels were assessed in the CML patient-derived cell line K562. Surprisingly, basal levels of RAS-GTP were low and JAK2 kinase activity was not detectable. Treatment of K562 cells with erythropoietin (EPO) only very modestly activated JAK2. Similarly, EPO treatment after one hour of BCR-ABL inhibition also failed to rescue STAT5 or RAS/ERK activation. However, with more prolonged BCR-ABL kinase inhibition, we observed a time-dependent increase in the ability of EPO to rescue STAT5 and ERK phosphorylation. After 24hrs of BCR-ABL inhibition, EPO treatment led to a significant increase in both JAK2 activation and the GTP loading of RAS relative to vehicle treated cells. These data suggest that BCR-ABL kinase activity hampers the ability of JAK2 to activate STAT5 and RAS/MAPK . Further, 24hr treatment with the MEK inhibitor PD0325901 allowed for a comparable degree of EPO-mediated JAK2 activation, suggesting that the negative regulation of JAK2 is mediated at least in part by a MEK/ERK-dependent mechanism. These observations were subsequently validated in an isogenic system utilizing TF-1 cells. Similar to the EPO/JAK2 axis in K562 cells, TF1-BCR-ABL cells exhibit attenuated GM-CSF signaling relative to control TF1-puro cells, which was restored only after prolonged BCR-ABL inhibition. Significantly, we have observed that BCR/ABL-expressing cells irreversibly commit to apoptosis before growth factor signaling is fully restored. Conclusions: Our data suggest that BCR-ABL-mediated oncogene addiction is a consequence of a high level of physiologic negative feedback mechanisms that dampen growth factor receptor signaling. Our findings also help reconcile recent evidence that JAK2 inhibition restores the sensitivity of CML stem/progenitor cells to ABL TKI treatment in vitro with the observation that JAK2 is completely dispensable for BCR-ABL-induced myeloid disease in mice. Citation Format: Jennifer Gajan, Elisabeth Lasater, Cheryl Tajon, Charles Craik, Neil Shah. A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4308. doi:10.1158/1538-7445.AM2013-4308

Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities.

Reactive oxygen species, such as superoxide and hydrogen peroxide, are generated in all cells by mitochondrial and enzymatic sources. Left unchecked, these reactive species can cause oxidative damage to DNA, proteins, and membrane lipids. Glutathione peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Certain reactive oxygen species, such as hydrogen peroxide, are also essential for growth factor-mediated signal transduction, mitochondrial function, and maintenance of normal thiol redox-balance. Thus, by limiting hydrogen peroxide accumulation, GPx-1 also modulates these processes. This review explores the molecular mechanisms involved in regulating the expression and function of GPx-1, with an emphasis on the role of GPx-1 in modulating cellular oxidant stress and redox-mediated responses. As a selenocysteine-containing enzyme, GPx-1 expression is subject to unique forms of regulation involving the trace mineral selenium and selenocysteine incorporation during translation. In addition, GPx-1 has been implicated in the development and prevention of many common and complex diseases, including cancer and cardiovascular disease. This review discusses the role of GPx-1 in these diseases and speculates on potential future therapies to harness the beneficial effects of this ubiquitous antioxidant enzyme.

Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists

Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC50 of 86μM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.

A new method for the purification of bioactive insulin-like growth factor-binding protein-3

We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3.

STAT5a/PPARγ Pathway Regulates Involucrin Expression in Keratinocyte Differentiation

Signal transducers and activators of transcription (STATs) are critical to growth factor-mediated intracellular signal transduction. We observed the rapid expression and activation of STAT5a during keratinocyte differentiation induced by suspension culture. STAT5a expression preceded that of involucrin, an important molecule in the terminal differentiation of keratinocytes. To determine whether STAT5a regulated involucrin expression, we expressed a dominant-negative (dn) STAT5a that blocks the dimerization of STAT5 and inhibits its nuclear translocation. We found that dn-STAT5a inhibited involucrin expression in keratinocytes. Given that STAT5 regulates adipogenesis via activating the peroxisome proliferator-activated receptor (PPAR) gamma signal, we hypothesized that STAT5a regulated involucrin expression in the same manner. To test this hypothesis, we examined the expression and transactivation of PPARgamma in a suspension culture of keratinocytes. Suspension culture induced PPARgamma expression and triggered PPARgamma transactivation rapidly and dn-STAT5a downregulated this induction and suppressed PPARgamma transactivation. Furthermore, preincubation with the PPARgamma/retinoid X-receptor inhibitor HX-531 or the introduction of a dn-PPARgamma prevented the activation of involucrin promoter and inhibited its induction. This report provides early evidence of a major role for STAT5a in the differentiation of keratinocytes, where it contributes to involucrin expression by activating the PPARgamma signal.

Molecular and Structural Basis for Redox Regulation of β-Actin

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H 2O 2. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H 2O 2 acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca 2+. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H 2O 2 in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H 2O 2. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.

APPL1 Associates with TrkA and GIPC1 and Is Required for Nerve Growth Factor-Mediated Signal Transduction

The neurotrophin receptor TrkA plays critical roles in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. Here, we report APPL1 as a TrkA-associated protein. APPL1 and TrkA co-immunoprecipitated in sympathetic neurons. We have identified two routes through which this association can occur. APPL1 was isolated as a binding partner for the TrkA-interacting protein GIPC1 from rat brain lysate by mass spectrometry. The PDZ domain of GIPC1 directly engaged the C-terminal sequence of APPL1. This interaction provides a means through which APPL1 may be recruited to TrkA. In addition, the APPL1 PTB domain bound to TrkA, indicating that APPL1 may associate with TrkA independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation determined that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that GIPC1 and APPL1 play a role in TrkA function and suggest that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals.

A Functional Interaction between Sprouty Proteins and Caveolin-1

Growth factor-mediated signal transduction cascades can be regulated spatio-temporally by signaling modulators, such as Sprouty proteins. The four mammalian Sprouty family members are palmitoylated phosphoproteins that can attenuate or potentiate numerous growth factor-induced signaling pathways. Previously, we have shown that Sprouty-1 and Sprouty-2 associate with Caveolin-1, the major structural protein of caveolae. Like Sprouty, Caveolin-1 inhibits growth factor-induced mitogen-activated protein kinase activation. Here, we demonstrate that all four mammalian Sprouty family members physically interact with Caveolin-1. The C terminus of Caveolin-1 is the major Sprouty-binding site, whereas Sprouty binds Caveolin-1 via its conserved C-terminal domain. A single point mutation in this domain results in loss of Caveolin-1 interaction. Moreover, we demonstrate that the various Sprouty isoforms differ dramatically in their cooperation with Caveolin-1-mediated inhibition of mitogen-activated protein kinase activation and that such cooperation is also highly dependent on the type of growth factor investigated and on cell density. Together, the data suggest that the Sprouty/Caveolin-1 interaction modulates signaling in a growth factor- and Sprouty isoform-specific manner.

Induction of Stem Cell Factor/c-Kit/Slug Signal Transduction in Multidrug-resistant Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is strongly resistant to conventional chemotherapy by unclear mechanisms. We and others have previously reported that cytokine- and growth factor-mediated signal transduction is involved in the growth and progression of MM. Here, we identified a pathway that involves stem cell factor (SCF)/c-Kit/Slug in mediating multidrug resistance of MM cells. When we compared gene expression profiles between five MM cells and their multidrug-resistant (MM DX) sublines, we found that MM DX cells expressed both SCF and c-Kit and had higher mRNA levels of Slug. Knockdown of c-Kit or Slug expression with their respective small interfering RNA sensitized MM DX cells to the induction of apoptosis by different chemotherapeutic agents, including doxorubicin, paclitaxel, and vincristine. Transfection of c-Kit in parental MM cells in the presence of SCF up-regulated Slug and increased resistance to the chemotherapeutic agents. Moreover, MM cells expressing Slug showed a similar increased resistance to the chemotherapeutic agents. These results indicate that induction of Slug by autocrine production of SCF and c-Kit activation plays a key role in conferring a broad spectrum chemoresistance on MM cells and reveal a novel signal transduction pathway for pharmacological or genetic intervention of MM patients.

An activated mTOR mutant supports growth factor-independent, nutrient-dependent cell survival.

In yeast, TOR couples cellular growth and metabolism to the availability of extracellular nutrients. In contrast, mammalian TOR kinase activity has been reported to be regulated by growth factor stimulation via the PI3K/Akt pathway. Consistent with this, growth factor deprivation results in dephosphorylation of the mTOR target proteins p70S6k and 4EBP1 in the face of abundant extracellular nutrients. To determine whether the activation of mTOR was sufficient to support cell survival in the absence of other growth factor-mediated signal transduction, we evaluated the ability of a growth factor-independent mTOR mutant, DeltaTOR, to protect cells from growth factor deprivation. DeltaTOR- but not wild-type mTOR-expressing cells were protected from many of the sequelae of growth factor deprivation including amino-acid transporter degradation, reduction of the glycolytic rate, cellular atrophy, decreased mitochondrial membrane potential, and Bax activation. Furthermore, DeltaTOR expression increased growth factor-independent, nutrient-dependent cell survival and enhanced the ability of p53-/- MEFs to form colonies in soft agar. These results suggest that activating mutations of mTOR can contribute to apoptotic resistance and might contribute to cellular transformation.

Recruitment and Activation of Phospholipase Cγ1 by Vascular Endothelial Growth Factor Receptor-2 Are Required for Tubulogenesis and Differentiation of Endothelial Cells

Vascular endothelial growth factor-mediated angiogenic signal transduction relay is achieved by coordinated induction of endothelial cell proliferation, migration, and differentiation. These complex cellular processes are most likely controlled by activation of both cooperative and antagonistic signals by vascular endothelial growth factor receptors (VEGFRs). Here, we investigated the contribution of tyrosine-phosphorylated residues of VEGFR-2/fetal liver kinase-1 to endothelial cell proliferation and differentiation and activation of signaling proteins. Mutation of tyrosine 1006 of VEGFR-2 to phenylalanine severely impaired the ability of this receptor to stimulate endothelial cell differentiation and tubulogenesis. Paradoxically, the mutant receptor stimulated endothelial cell proliferation far better than the wild-type receptor. Further analysis showed that tyrosine 1006 is responsible for phospholipase Cgamma1 (PLCgamma1) activation and intracellular calcium release in endothelial cells. Activation of PLCgamma1 was selectively mediated by tyrosine 1006. Mutation of tyrosines 799, 820, 949, 994, 1080, 1173, and 1221 had no measurable effect on the ability of VEGFR-2 to stimulate PLCgamma1 activation. Association of VEGFR-2 with PLCgamma1 was mainly established between tyrosine 1006 and the C-terminal SH2 domain of PLCgamma1 in vitro and in vivo. Taken together, the results indicate that phosphorylation of tyrosine 1006 is essential for VEGFR-2-mediated PLCgamma1 activation, calcium flux, and cell differentiation. More importantly, VEGFR-2-mediated endothelial cell proliferation is inversely correlated with the ability of VEGFR-2 to associate with and activate PLCgamma1.

Sirolimus and islet transplants

IMMUNOSUPPRESSION is a critical determinant of the success of islet transplants. Tailoring immunosuppression to the specific setting of marginal mass islet cell transplants in autoimmune type 1 diabetic recipients would likely increase the safety, success rate, and applicability of such transplants. Tailored immunosuppression would ideally inhibit antigen-independent innate immune responses during the engraftment of transplanted islets, control alloimmune responses, and thwart autoimmune responses without diabetogenic and nephrotoxic side effects. Rejectionfree protocols need to be administered until markers of imminent islet rejection are validated or until robust function of a dysfunctional graft is restored. Sirolimus is a macrocyclic lactone with potent immunosuppressive properties. Its mechanism of action involves the blockade of IL-2 and other T-cell growth factor-mediated signal transduction and the inhibition of cell cycle progression. Sirolimus is conceivably the single most important contribution to the immunosuppressive armamentarium in islet transplantation. The immunosuppressive synergism between sirolimus and cyclosporin A (CsA) markedly reduces the incidence of solid organ and islet allograft rejection; it also enables steroids to be avoided and CsA doses to be profoundly reduced. In vitro studies suggested competitive inhibition between sirolimus and tacrolimus. Synergism between those two drugs was demonstrated in rat transplant models. The synergism between sirolimus (and other mammalian targets of rapamycin antagonists) and sphingosine 1-phosphate receptor agonists enables both steroids and calcineurin inhibitors to be avoided, thereby protecting allografts from rejection as well as from diabetogenic and nephrotoxic side effects. The synergism between sirolimus and selective blockers of costimulatory pathways helps induce immunologic tolerance and may, in the future, facilitate minimization or discontinuation of maintenance immunosuppression. The distinctive profile of sirolimus suggests an important role for it as an immunosuppressive and tolerogenic agent for diabetic recipients of islet transplants. Studies of the tolerogenic potential of sirolimus have been performed predominantly in cardiac and islet transplant models in mice. Sirolimus was used at a dose of 0.2 mg/kg/d intraperitoneally (IP) daily for the first 3 days, then every other day until the advent of hyperglycemia. It markedly prolonged the survival of islet allografts across a complete major histocompatibility barrier in interleukin (IL)-2 / and IL-4 / double knockout mice. Thus, sirolimus blocks T-cell growth factor signals beyond IL-2 and IL-4, especially IL-7 and IL-15; these rapamycin-sensitive T-cell growth factor signals are critical in the execution of islet allograft rejection. Combined costimulatory blockade (anti-CD154 monoclonal antibody and CTLA4-Ig) and sirolimus monotherapy 0.2 mg/kg/d IP on days 0, 1, 2, then every other day through day 14 posttransplant both facilitated indefinite survival of cardiac allografts in the Balb/c to C3H/He strain combination. Adding CsA, but not sirolimus, to treatment with anti-CD154 monoclonal antibody and CTLA4-Ig blocked the capacity of costimulatory blockade to produce permanent engraftment. Subsequently, islet transplant experiments were conducted in IL-2-deficient mice treated with sirolimus, and cardiac allograft transplant experiments in Bcl-XL transgenic mice treated with costimulatory blockade. Those experiments assessed the importance of alloreactive T-cell pool size, and more specifically activated-induced cell death of alloreactive T cells (in peripheral tolerance induction). They confirmed the hypothesis that sirolimus blocks the proliferative component of IL-2 signaling but does not inhibit priming of activation-induced cell death. They also demonstrated that deletion of activated T cells, through activation-induced cell death or growth factor withdrawal, is necessary to achieve peripheral tolerance across major histocompatibility barriers. The efficacy of sirolimus in preventing spontaneous and recurrent autoimmune diabetes has been studied in NOD mice, a clinically relevant model of autoimmune type 1 diabetes. Sirolimus was administered per os (PO) 3 times per week from 8 to 10 weeks of age; it prevented onset of autoimmune diabetes in 90% of NOD mice at a dose of 0.6

Critical Roles of Thioredoxin in Nerve Growth Factor-Mediated Signal Transduction and Neurite Outgrowth in PC12 Cells

Thioredoxin (TRX) has a role in a variety of biological processes, including cytoprotection and the activation of transcription factors. Nerve growth factor (NGF) is a major survival factor of sympathetic neurons and promotes neurite outgrowth in rat pheochromocytoma PC12 cells. In this study, we showed that NGF induces TRX expression at protein and mRNA levels. NGF activated the TRX gene through a regulatory region positioned from -263 to -217 bp, containing the cAMP-responsive element (CRE). Insertion of a mutation in the CRE in this region abolished the response to NGF. NGF induced binding of CRE-binding protein to the CRE of the TRX promoter in an electrophoretic mobility shift assay. NGF also induced nuclear translocation of TRX. 2'-Amino-3'-methoxyflavone, an inhibitor of mitogen-activated protein kinase kinase, which is a known inhibitor of NGF-dependent differentiation in PC12 cells, suppressed the NGF-dependent expression and nuclear translocation of TRX. Overexpression of mutant TRX (32S/35S) or TRX antisense vector blocked the neurite outgrowth of PC12 cells by NGF. Overexpression of mutant TRX (C32S/C35S) suppressed the NGF-dependent activation of the CRE-mediated c-fos reporter gene. These results suggest that TRX plays a critical regulatory role in NGF-mediated signal transduction and outgrowth in PC12 cells.

A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction.

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways. Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling. We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation. The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2. Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E. Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade. These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment.

Growth factor-mediated signal transduction and redox balance in isolated digestive gland cells from Mytilus galloprovincialis Lam.

In mammalian cells, a growing body of evidence indicates a relationship between cellular redox balance and tyrosine kinase-mediated cell signalling. The phosphorylative cascade activated by extracellular signals is inhibited by reducing conditions and stimulated by oxidative stress, in particular at the level of mitogen activated protein kinase (MAPK) activation. The mussel Mytilus typically shows variations in antioxidant defence systems and decreases in glutathione content in response to both natural and contaminant environmental stressors. In isolated mussel digestive gland cells, both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) have been recently demonstrated to activate tyrosine kinase receptors leading to multiple responses; among these, stimulation of the key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). The present study investigates the possible relationship between the tyrosine kinase-mediated metabolic effects of growth factors and cellular redox balance in mussel cells. The results demonstrate that the effects of growth factors on glycolytic enzymes were abolished by cell pretreatment with the antioxidant N-acetyl-cysteine (NAC). On the other hand, in cells where the glutathione content and synthesis were lowered either in vitro (by cell pretreatment with buthionine sulfoximine (BSO)), or in vivo (by mussel exposure to Cu 2+) the metabolic effects of growth factors were unaffected. Moreover, the results show that, in both control and glutathione-depleted cells, growth factors can also regulate the level of glutathione apparently by modulating, via phosphorylative mechanisms involving MAPK activation, the activity of γ-glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. Overall, this study extends the hypothesis that cell signalling is intimately related to redox balance in marine invertebrate cells.

The human papillomavirus type 16 E5 protein modulates phospholipase C-gamma-1 activity and phosphatidyl inositol turnover in mouse fibroblasts.

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-gamma-1 (PLC-gamma-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-gamma-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.