Dependent Signal Transduction Research Articles

COMMD1 is an Immune Regulatory Gene That Plays a Role In IBD Pathogenesis

The 2p15 locus has been linked to risk for inflammatory disorders such as ankylosing spondylitis, and more recently, Crohn’s disease. However, the precise gene in this region that is responsible for these associations remains uncertain. A metaanalysis of GWAS data as well eQTL analysis was employed to ascertain potential candidate genes in the 2p15 locus. The biologic relevance of the presumed associations were then analyzed utilizing conditional gene inactivation in mice. We identified a specific single nucleotide polymorphism (SNP) in the 2p15 region that was linked to higher risk for ulcerative colitis. This SNP was located just 3′ of the COMMD1 gene and eQTL analysis in 2 different cohorts indicated that this SNP correlates with differential expression of COMMD1 mRNA in normal subjects. To further examine the potential role of COMMD1 in IBD pathogenesis, we employed murine models of conditional gene inactivation. We show that Commd1 loss in the myeloid lineage, a central arm of the innate immune system, leads to exaggerated inflammatory responses due to decreased termination of NF-kB initiated gene expression. Moreover, these animals demonstrate an increased predisposition to colitis in a variety of models, including DSS-induced colitis and IL-10 deficiency. In addition to greater inflammation, an increased progression to dysplasia and frank neoplasia was also evident. Finally, we find that mucosal inflammation in IBD patients leads to decreased COMMD1 expression. This suppression of COMMD1, which is pro-inflammatory, is mediated by IFN-gamma dependent signal transduction pathways that affect COMMD1 mRNA stability. Altogether, our studies demonstrate that the COMMD1 gene plays an anti-inflammatory role and that decreased expression plays a role in the pathogenesis of IBD.

Effectiveness and Safety of a Therapeutic Vaccine Against Angiotensin II Receptor Type 1 in Hypertensive Animals

Primary hypertension is a chronic disease with high morbidity, and the rate of controlled blood pressure is far from satisfactory, worldwide. Vaccination provides a promising approach for treatment of hypertension and improvement in compliance. Here, the ATRQβ-001 vaccine, a peptide (ATR-001) derived from human angiotensin II (Ang II) receptor type 1 conjugated with Qβ bacteriophage virus-like particles, was developed and evaluated in animal models of hypertension. The ATRQβ-001 vaccine significantly decreased the blood pressure of Ang II-induced hypertensive mice up to 35 mm Hg (143 ± 4 versus 178 ± 6 mm Hg; P=0.005) and that of spontaneously hypertensive rats up to 19 mm Hg (173 ± 2 versus 192 ± 3 mm Hg; P=0.003) and prevented remodeling of vulnerable hypertensive target organs. No obvious feedback activation of circulating or local renin-angiotensin system was observed. Additionally, no significant immune-mediated damage was detected in vaccinated hypertensive and nonhypertensive animals. The half-life of the anti-ATR-001 antibody was 14.4 days, surpassing that of existing chemical drugs. In vitro, the anti-ATR-001 antibody specifically bound to Ang II receptor type 1 and inhibited Ca(2+)-dependent signal transduction events, including protein kinase C-α translocation, extracellular signal-regulated kinase 1/2 phosphorylation (72% decrease; P=0.013), and elevation of intracellular Ca(2+) (68% decrease; P=0.017) induced by Ang II, but without inhibiting Ang II binding to the receptor. In conclusion, the ATRQβ-001 vaccine decreased the blood pressure of Ang II-induced hypertensive mice and spontaneously hypertensive rats effectively through diminishing the pressure response and inhibiting signal transduction initiated by Ang II. Thus, the ATRQβ-001 vaccine may provide a novel and promising method for the treatment of primary hypertension.

Venous thromboembolism and coagulation activity in patients with immune thrombocytopenia treated with thrombopoietin receptor agonists

Venous thromboembolism and coagulation activity in patients with immune thrombocytopenia treated with thrombopoietin receptor agonists

HLA Class I: An unexpected role in integrin β4 signaling in endothelial cells

The production of anti-donor antibodies to HLA class I and class II antigens following transplantation is associated with development of transplant vasculopathy and graft loss. Antibodies against HLA class I (HLA-I) molecules are thought to contribute to transplant vasculopathy by triggering signals that elicit the activation and proliferation of endothelial cells. The proximal molecular events that regulate HLA-I dependent signal transduction are not well understood. We demonstrated a mutual dependency between HLA-I and integrin β4 to stimulate signal transduction and cell proliferation. Similarly, we found that integrin β4-mediated cell migration was dependent upon its interactions with HLA-I molecules. Since integrin β4 has been implicated in angiogenesis and tumor formation, associations between integrin β4 and HLA-I may play an important role in cancer. Further characterization of interactions between HLA-I and integrin β4 may lead to the development of therapeutic strategies for the treatment and prevention of chronic allograft rejection and cancer.

Abstract 3803: Anti-cancer activity of Capridine, a novel nitroacridine chemotherapeutic, in glioblastoma

Abstract Glioblastoma is characterized by highly proliferative tumor cells, increased cellularity, necrosis, and proliferation of capillary endothelial cells. The incidence rate of glioma in adults is 50% greater in men than in women suggesting that there may be a male hormonal component that modulates glioblastoma progression. Our preclinical developmental program identified Capridine as a chemotherapeutic agent that had specificity towards prostate cancer with minimal bone marrow toxicity. Since the DNA intercalating activity of Capridine does not completely correlate with its cytotoxic activity, we examined other alternative cellular targets. Our investigation identified the androgen receptor (AR) and its dependent signal transduction mechanism as a possible cellular mediator of its cytotoxic activity. In this study, our analysis revealed that glioblastoma cell lines T98G and MO59K expressed AR and their growth was responsive to the AR-mediated signal transduction. Capridine inhibited the growth of glioblastoma cells by down regulating the AR and inhibiting the phosphorylation of Focal adhesion kinase (FAK). Since FAK is associated with integrin and neuropeptide mediated signaling, critical for cell migration and invasion, we examined the activity of Capridine on cell invasion and migration using a Boyden Chamber assay. Capridine inhibited the migration and invasion of the glioblastoma cell lines T98G and MO59K. Our data not only validate AR as a target of Capridine as with prostate cancer but also implicate a novel target, FAK in glioblastoma. Combination approaches using Capridine and anti-hormonal therapy may prove to be viable chemotherapeutic regimen for primary glioblastoma as well as an inhibitor of metastatic propensity. These preclinical studies are being extended in an in vivo animal model with combination treatment approaches with a view to develop novel chemotherapy based treatment for glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3803. doi:1538-7445.AM2012-3803

Abstract 3980: Computational analysis of paclitaxel-induced tumor priming that leads to enhanced deposition and efficacy nanoparticulate doxorubicin

Abstract Purpose: Paclitaxel (PAC) has been shown to cause tumor priming (TP), which increases delivery of subsequently-administered drugs. A wave of PAC-mediated apoptosis results in a transient reduction in tumor cell density, expansion of the interstitial space, and vascular permeability compromise that enhances tumor deposition of nanoparticulate formulations such as liposomal doxorubicin (L-dox). Our aim was to develop an integrated quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis approach to optimize combination chemotherapy of PAC with L-dox. Methods: Data were extracted from the study of Lu et al. (J Pharmacol Exp Ther 322: 80, 2007) in which mice bearing human tumor xenografts were treated with PAC (40 mg/kg), L-dox (20 mg/kg), TP dosing (PAC followed by L-dox at 48h, the peak of the priming effect), or the reverse sequence (L-dox then PAC). Data included plasma and tumor drug concentrations, kinetics of PAC and L-dox induced apoptosis, and % change in tumor volume from baseline. Drug profiles were described with a PK model for carrier-mediated PAC, and a two-compartment model for L-dox, with first-order release of free drug from the liposome. Intratumor concentrations were described using a hybrid well-stirred model with estimated partition coefficients (kp) for each drug. Tumor PK profiles were used to drive apoptotic responses, which were modeled using nonlinear time dependent signal transduction functions. The tumor growth PD model consisted of two transit compartments, with a first order net growth rate constant and the apoptotic signals from each agent driving cytotoxic effects. PAC induced TP was modeled using a feedback loop, with the apoptosis signal of PAC enhancing the deposition of L-dox (i.e. increasing kp for dox). Model fittings were performed using MATLAB, and simulations were conducted to explore priming sequences. Results: The final model captured well the PK of PAC and L-dox in plasma and tumors, and the time course of apoptosis induction, and tumor growth for each treatment sequence. With single agent dosing, kp values for PAC and L-dox were estimated at 0.044 and 0.085. L-dox kp increased 2-fold after the TP treatment. Apoptosis signals exhibited a delayed onset that was well captured, and the intratumor concentrations producing maximum effects (Emax) and 50% Emax were 18 and 7.2 μg/mL (PAC) and 17.6 and 14.3 μg/mL (L-dox). The duration of drug induced apoptosis was 27.4 h for PAC and 15.8 h for L-dox. Simulation with the PK/PD model suggested that earlier administration of L-dox would increase efficacy markedly. Conclusions: A model was successfully developed that captured the priming effect on PK and efficacy. Simulation suggested that administration of L-dox 24 h prior to the priming peak would enhance efficacy further. This model could be adapted for evaluating other combination chemotherapies using PAC as a TP agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3980. doi:1538-7445.AM2012-3980

Stress‐activated cellular senescence as a mechanism of melanocytes degeneration in vitiligo

Several studies show evidence of an alterated redox status and an increased susceptibility of vitiligo cells to pro‐oxidant agents, supporting the idea that an imbalance of the cellular redox status might be the pathogenic mechanism in vitiligo. In order to study intracellular pathways involved in stress‐dependent vitiligo cell damage, we focused on cAMP/PKA/CREB and mitogen activated protein kinases (MAPK)‐dependent signal transduction. We found correlation between intrinsic oxidative stress, activation MAPK and p53, hyperphosphorylation of CREB and increased cholesterol levels. Notably, these long‐term effects of subcytotoxic oxidative stress are also biomarkers of premature cellular senescence. Consistent with this, vitiligo cells showed a significant increase in p16 that did not correlate with the chronological age of the donors. Chronic stimulation of MAPK and downstream inhibition of cyclin‐dependent kinases in the presence of elevated levels of cyclinD1 suggest that vitiligo melanocytes are prone to entering a hypermitogenic cell cycle arrest. Accordingly, vitiligo cells displayed attenuated responses to mitogens. Moreover, vitiligo melanocytes produced biologically active proteins among the senescence‐associated secretory phenotype, such as MMP3, Cox‐2, IGFBPs. Together, these data argue for a pathologic predisposition of vitiligo cells to stress‐induced premature senescence.

BioKnife, a uPA Activity-dependent Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma via Simultaneous Stimulation of uPA Expression

Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.

Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideumcells

BackgroundCell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development.ResultsHere, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in aca- cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels.ConclusionsCollectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.

Dynamic model of IL–6 and IL–1beta dependent signalling in primary murine hepatocytes

The hepatic inflammatory response is an important part of the host defence and also represents a critical regulatory event in hepatic liver regeneration. It is mainly orchestrated by a mediator based communication between the different cell types in the liver, including parenchymal cells and non-parenchymal cells, such as sessile liver macrophages (Kupffer cells). In this context, a cytokine mediated signal always depends on the background of co-acting factors and the type of target cells. In macrophages the interleukin–6 (IL–6) induced STAT3 (signal transducer and activator of transcription) activation is abrogated in the presence of LPS (lipopolysaccharide) or TNF-α (tumor necrosis factor), whereas in hepatocytes the STAT3 dependent signal transduction of IL–6 is abolished in the presence of IL–1β. This inhibitory effect is largely mediated by IL–1β dependent activation of the p38MAPK cascade.

Blend of Roselle Calyx and Selected Fruit Modulates Testicular Redox Status and Sperm Quality of Diabetic Rats

Mammalian fertilization, a species-specific event, is the net result of a highly orchestrated process that collectively results in the fusion of two radically different-looking haploid cells, sperm and egg, to form a diploid zygote, a cell with somatic chromosome numbers. Prior to the interaction of the opposite gametes, mammalian spermatozoa undergo many fascinating changes during development in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only the capacitated spermatozoa interact with the extracellular coat, the zona pellucida, which surrounds the mammalian oocyte. The tight and irreversible binding of the opposite gametes in the mouse and many other mammals studied, including human, starts a ca2+- dependent signal transduction pathway that results in the exocytosis of acrosomal contents at the site of the sperm binding. The hydrolytic action of the acrosomal glycohydrolases and proteinases, released at the site of the sperm-egg binding, along with the enhanced thrust generated by the hyperactivated spermatozoon, are important factors that regulate the penetration of the zona pellucida and fusion of the oppositegametes. The purpose of this editorial is to highlight the well programmed molecular events that are necessary before sperm-egg adhesion. In addition, my intention is to discuss the increasing controversy about the mechanism(s) that regulate mammalian sperm-egg interactions.

Biological Processes that Prepare Mammalian Spermatozoa to Interact with an Egg and Fertilize It

In the mouse and other mammals studied, including man, ejaculated spermatozoa cannot immediately fertilize an egg. They require a certain period of residence in the female genital tract to become functionally competent cells. As spermatozoa traverse through the female genital tract, they undergo multiple biochemical and physiological changes collectively referred to as capacitation. Only capacitated spermatozoa interact with the extracellular egg coat, the zona pellucida. The tight irreversible binding of the opposite gametes triggers a Ca2+-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane and the underlying outer acrosomal membrane at multiple sites that causes the release of acrosomal contents at the site of sperm-egg adhesion. The hydrolytic action of the acrosomal enzymes released, along with the hyperactivated beat pattern of the bound spermatozoon, is important factor that directs the sperm to penetrate the egg coat and fertilize the egg. The sperm capacitation and the induction of the acrosomal reaction are Ca2+-dependent signaling events that have been of wide interest to reproductive biologists for over half a century. In this paper, we intend to discuss data from this and other laboratories that highlight the biological processes which prepare spermatozoa to interact with an egg and fertilize it.

Mechanisms of Mammalian Sperm-Egg Interaction Leading to Fertilization

Mammalian fertilization, a species-specific event, is the net result of a highly orchestrated process that collectively results in the fusion of two radically different-looking haploid cells, sperm and egg, to form a diploid zygote, a cell with somatic chromosome numbers. Prior to the interaction of the opposite gametes, mammalian spermatozoa undergo many fascinating changes during development in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only the capacitated spermatozoa interact with the extracellular coat, the zona pellucida, which surrounds the mammalian oocyte. The tight and irreversible binding of the opposite gametes in the mouse and many other mammals studied, including human, starts a ca2+- dependent signal transduction pathway that results in the exocytosis of acrosomal contents at the site of the sperm binding. The hydrolytic action of the acrosomal glycohydrolases and proteinases, released at the site of the sperm-egg binding, along with the enhanced thrust generated by the hyperactivated spermatozoon, are important factors that regulate the penetration of the zona pellucida and fusion of the oppositegametes. The purpose of this editorial is to highlight the well programmed molecular events that are necessary before sperm-egg adhesion. In addition, my intention is to discuss the increasing controversy about the mechanism(s) that regulate mammalian sperm-egg interactions.

<i>In vivo</i> effects of genistein, herbimycin a and geldanamycin on rat hepatic cytochrome P4501A

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) are regulated through the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway and are generally known as enzymes which metabolize anthropogenic xenobiotics such as dioxin to carcinogenic and mutagenic compounds. However, recently the facts of CYP1A activation under physiological conditions or under action of non-dioxin-like compounds appear. In the present study we show that genistein, herbimycin A and geldanamycin (the protein-tyrosine kinase inhibitors) affect in vivo to CYP1A1 activity, the CYP1A1 mRNA level and the CYP1A1 protein level. These data provide insight into the role of protein kinases in CYP1A regulation may facilitate the understanding of CYP1A regulation.

An Important Role for Rac1 in Glycoprotein Ib-IX Induced Platelet Activation,

Abstract 3250The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, not only plays a critical role in mediating platelet adhesion at sites of vascular injury, but also transmits signals leading to platelet activation. GPIb-IX mediated activation of the ligand binding function of integrin αIIbβ3 is required for stable platelet adhesion under high shear rate conditions. Previous studies have suggested that ligand occupancy of GPIb-IX leads to activation of the Src family kinase Lyn, phosphoinositide 3-kinase (PI3K), Akt, cGMP-dependent protein kinase, and the MAPK and ITAM signaling pathways. However, it remains unclear how the PI3K/Akt pathway is activated during GPIb-IX dependent signal transduction. Using platelet-specific conditional Rac1−/− mice and the Rac1 specific inhibitor NSC23766 (NSC), we examined the role of Rac1 in GPIb-IX induced platelet activation. Platelet-specific conditional Rac1−/− mice were generated by crossing Pf4-Cre transgenic mice with mice containing the Rac1 conditional allele. Rac1−/− mouse platelets had a defect in VWF/botrocetin induced aggregation, TXA2 synthesis, and ATP secretion. Rac1−/− mouse platelets were also defective in stable adhesion to VWF under shear stress and in spreading on immobilized VWF, as compared to WT. The defects observed in Rac1−/− mouse platelets were recapitulated in human platelets treated with the Rac1 inhibitor NSC23766. Importantly, we show that ligand binding to GPIb-IX induced Rac1 activation, which was abolished in Lyn−/− mouse platelets. We also show that Rac1−/− (or NSC-treated) platelets have a defect in GPIb-IX induced Akt and P38 MAPK activation. Altogether, this study reveals that Rac1 plays an important role in GPIb-IX dependent platelet activation and stable adhesion to VWF under shear stress. Rac1 is activated downstream of Lyn and is important for GPIb-IX and Lyn-dependent activation of Akt and MAPK signaling pathways. Disclosures:No relevant conflicts of interest to declare.

PINK1 enhances insulin-like growth factor-1-dependent Akt signaling and protection against apoptosis

Mutations in the PARK6 gene coding for PTEN-induced kinase 1 (PINK1) cause recessive early-onset Parkinsonism. Although PINK1 and Parkin promote the degradation of depolarized mitochondria in cultured cells, little is known about changes in signaling pathways that may additionally contribute to dopamine neuron loss in recessive Parkinsonism. Accumulating evidence implicates impaired Akt cell survival signaling in sporadic and familial PD (PD). IGF-1/Akt signaling inhibits dopamine neuron loss in several animal models of PD and both IGF-1 and insulin are neuroprotective in various settings. Here, we tested whether PINK1 is required for insulin-like growth factor 1 (IGF-1) and insulin dependent phosphorylation of Akt and the regulation of downstream Akt target proteins. Our results show that embryonic fibroblasts from PINK1-deficient mice display significantly reduced Akt phosphorylation in response to both IGF-1 and insulin. Moreover, phosphorylation of glycogen synthase kinase-3β (GSK-3β) and nuclear exclusion of FoxO1 are decreased in IGF-1 treated PINK1-deficient cells. In addition, phosphorylation of ribosomal protein S6 is reduced indicating decreased activity of mitochondrial target of rapamycin (mTOR) in IGF-1 treated PINK1−/− cells. Importantly, the protection afforded by IGF-1 against staurosporine-induced metabolic dysfunction and apoptosis is abrogated in PINK1-deficient cells. Moreover, IGF-1-induced Akt phosphorylation is impaired in primary cortical neurons from PINK1-deficient mice. Inhibition of cellular Ser/Thr phosphatases did not increase the amount of phosphorylated Akt in PINK1−/− cells, suggesting that components upstream of Akt phosphorylation are compromised in PINK1-deficient cells. Our studies show that PINK1 is required for optimal IGF-1 and insulin dependent Akt signal transduction, and raise the possibility that impaired IGF-1/Akt signaling is involved in PINK1-related Parkinsonism by increasing the vulnerability of dopaminergic neurons to stress-induced cell death.

Socking It to Cardiac Hypertrophy

In the heart, increases in contractile load provoked by hypertension or valvular disease promote increases in cardiac mass through the hypertrophy of cardiomyocytes. Injury or death to cardiomyocytes in a portion of the heart places a greater load on cells within uninjured areas and stimulates cellular hypertrophy. Similarly, in familial forms of hypertrophic cardiomyopathy, defects in genes encoding proteins of the sarcomere promote growth in the absence of a stimulus. Hypertrophic hearts are susceptible to abnormalities of cardiac rhythm and have impaired relaxation (diastolic dysfunction), although contractile performance can be preserved. However, when hypertrophic stimuli are unrelieved, the remodeling phenomenon progresses and increasing numbers of cardiomyocytes are replaced with fibrotic scar, which leads to decreased systolic function and circulatory failure. Article see p 796 Neurotransmitters or hormones that bind to cell surface receptors modulate the rate and force of contraction by regulating the flux of calcium across the cell membrane or released from the sarcoplasmic/endoplasmic reticulum (SR/ER). In addition, Ca2+ signaling in the cardiomyocyte regulates the long-term remodeling responses of the heart to changing workloads by altering Ca2+-dependent gene expression and metabolism.1 In this way, Ca2+ can sense and respond to changes in muscle contraction through a link with hypertrophic gene expression.2 It is not intuitively obvious, however, how these Ca2+ signals could serve simultaneously as the proximate signal to control cardiac mass, specialized programs of gene expression, and the contractile and metabolic capacities of cardiomyocytes. Ca2+-dependent signaling events activated by increased contractile load somehow must be insensitive to the large fluctuations in cytosolic calcium that occur as a function of ambient contractile activity. This consideration may explain why although Ca2+-dependent signal transduction pathways that modulate gene expression have been well defined in other cell types for many years, …

Attachment of HeLa cells during early G1 phase

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.

Opposing effects of cortisol and dehydroepiandrosterone on the expression of the receptor for Activated C Kinase 1: Implications in immunosenescence

Aging is associated to a decline in immune functions that are in part related to a defective protein kinase C dependent signal transduction machinery. RACK-1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. We have previously demonstrated, in the elderly, a defective PCKβII (Protein Kinase C βII) translocation related to a decrease in RACK-1 protein expression, which is correlated to the age-associated decline in DHEA (dehydroepiandrosterone) levels. As a consequence of this signal transduction impairment, a significant decrease in immune cells functionality was observed. Furthermore, we could demonstrate that in vivo and in vitro DHEA administration restored RACK-1 level and immune functions, indicating that this hormone behaved as a positive RACK-1 regulator. We have most recently characterized the human GNB2L1 promoter region, coding for RACK-1 protein. Although no direct DHEA responsive elements were found, a glucocorticoid responsive element (GRE) was identified. The purpose of this work was to investigate, in the human pro-myelocytic cell line THP-1, whether physiological cortisol concentrations were able to modulate GNB2L1 promoter activity, RACK-1 transcription as well as cytokine production. As DHEA is endowed of anti-glucocorticoid properties in several cellular systems, and as cortisol:DHEA ratio imbalance is relevant in aging, we also investigated their possible interaction at the RACK-1 expression level. We could demonstrate that cortisol acted in a dose-related manner as a GNB2L1 promoter repressor, reducing RACK-1 mRNA expression and protein level. Probably by interfering with glucocorticoid receptor binding to GRE sequence, prolonged DHEA exposure counteracted cortisol effects, restoring RACK-1 levels and cytokine production, as assessed by LPS-induced TNF-α release.

Toll-like receptor 4-mediated cAMP production up-regulates B-cell activating factor expression in Raw264.7 macrophages

B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression.