Post-receptor Signaling Events Research Articles

Agonist-induced desensitisation of β3 -adrenoceptors: Where, when, and how?

β3 -Adrenoceptor agonists have proven useful in the treatment of overactive bladder syndrome, but it is not known whether their efficacy during chronic administration may be limited by receptor-induced desensitisation. Whereas the β2 -adrenoceptor has phosphorylation sites that are important for desensitisation, the β3 -adrenoceptor lacks these; therefore, it had been assumed that β3 -adrenoceptors are largely resistant to agonist-induced desensitisation. While all direct comparative studies demonstrate that β3 -adrenoceptors are less susceptible to desensitisation than β2 -adrenoceptors, desensitisation of β3 -adrenoceptors has been observed in many models and treatment settings. Chimeric β2 - and β3 -adrenoceptors have demonstrated that the C-terminal tail of the receptor plays an important role in the relative resistance to desensitisation but is not the only relevant factor. While the evidence from some models, such as transfected CHO cells, is inconsistent, it appears that desensitisation is observed more often after long-term (hours to days) than short-term (minutes to hours) agonist exposure. When it occurs, desensitisation of β3 -adrenoceptors can involve multiple levels including down-regulation of its mRNA and the receptor protein and alterations in post-receptor signalling events. The relative contributions of these mechanistic factors apparently depend on the cell type under investigation. Which if any of these factors is applicable to the human urinary bladder remains to be determined. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Flt3 Ligand Induces Monocyte Proliferation and Enhances the Function of Monocyte-Derived Dendritic Cells In Vitro

Flt3 ligand (FL), a potent hematopoietic cytokine, plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). Although some post-receptor signaling events of FL have been characterized, the role of FL on Flt3 expressing human peripheral blood monocyte is unclear. In the current study, we examined the role of FL on cell survival and growth of peripheral blood monocytes and function of monocyte-derived DCs. FL promoted monocyte proliferation in a dose-dependent manner and prevented spontaneous apoptosis. FL induced ERK phosphorylation and a specific ERK inhibitor completely abrogated FL-mediated cellular growth, while p38 MAPK, JNK, and AKT were relatively unaffected. Addition of FL to GM-CSF and IL-4 during DCs generation from monocytes increased the yield of DCs through induction of cell proliferation. DCs generated in the presence of FL expressed more costimulatory molecules on their surfaces and stimulated allogeneic T cell proliferation in MLR to a higher magnitude. Furthermore, FL partially antagonized IL-10-mediated inhibition on DCs function. Further characterization of FL actions may provide new and important information for immunotherapeutic approaches utilizing DCs.

Adiponectin as a potential biomarker of vascular disease

The increasing prevalence of diabetes and its complications heralds an alarming situation worldwide. Obesity-associated changes in circulating adiponectin concentrations have the capacity to predict insulin sensitivity and are a link between obesity and a number of vascular diseases. One obvious consequence of obesity is a decrease in circulating levels of adiponectin, which are associated with cardiovascular disorders and associated vascular comorbidities. Human and animal studies have demonstrated decreased adiponectin to be an independent risk factor for cardiovascular disease. However, in animal studies, increased circulating adiponectin alleviates obesity-induced endothelial dysfunction and hypertension, and also prevents atherosclerosis, myocardial infarction, and diabetic cardiac tissue disorders. Further, metabolism of a number of foods and medications are affected by induction of adiponectin. Adiponectin has beneficial effects on cardiovascular cells via its antidiabetic, anti-inflammatory, antioxidant, antiapoptotic, antiatherogenic, vasodilatory, and antithrombotic activity, and consequently has a favorable effect on cardiac and vascular health. Understanding the molecular mechanisms underlying the regulation of adiponectin secretion and signaling is critical for designing new therapeutic strategies. This review summarizes the recent evidence for the physiological role and clinical significance of adiponectin in vascular health, identification of the receptor and post-receptor signaling events related to the protective effects of the adiponectin system on vascular compartments, and its potential use as a target for therapeutic intervention in vascular disease.

Profound Signaling Abnormalities Due to Cytoplasmic Variants of CSF3R in Patients with Myelodysplastic/Myeloproliferative Syndromes Associated with CSF3R Mutations or Mutations of Splicing Factor U2AF1

AbstractAbstract 3792Myelodysplastic Syndromes (MDS) are a group of bone marrow disorders closely related to acute myeloid leukemia (AML). Even though a number of genetic mutations have been recently identified in patients with MDS, their contributions to MDS pathogenesis remains poorly understood. Some of these genetic mutations involve transcription factors, but they are also found in AML: TET2, EZH2, and ASXL1. One group of mutations distinct to MDS are those encoding proteins involved in RNA splicing (e.g. U2AF1/U2AF35, ZRSR2, SRSF2, SF3B1). Based on RNA-Seq of MDS/AML patients, we report exon skipping in the 3′ end of the CSF3R transcript, which encodes the granulocyte colony-stimulating factor receptor (GCSFR), in a patient carrying the S34F mutation in the U2AF1 gene. U2AF1 is one of the more recurrent genes affected by mutation in MDS, and it is associated with progression to secondary AML. The S34F mutation in U2AF1 is a gain of function mutation that promotes excess splicing and exon skipping. Alternative splicing of CSF3R results in 7 transcripts, of which the two most common are Class I and Class IV. There are putative splicing sequences within exon 17 of the CSF3R locus; GT (GU) at the 5′ site and AG at the 3′ site - a recognition sequence for U2AF1. In addition, we also identified mutations affecting CSF3R in two patients with chronic myelomonocytic leukemia (GCSFR T595I or Q726X), one patient with Refractory Cytopenias with Multilineage Dysplasia and Ring Sideroblasts (GCSFR W650L), and one patient with primary AML (GCSFR G659fs). In the last case, this mutation affects the Class III transcript of CSF3R, an alternatively spliced form expressed highly in the placenta. The Class IV isoform lacks much of the C-terminal domain, similar to the protein produced by nonsense mutations found in patients with severe congenital neutropenia who develop MDS/AML or the patient we identified.Little is known about the signaling-phenotype relationship of a mutant or alternatively-spliced GCSFR. To address these questions, we first studied the expression patterns of Class I and Class IV GCSFR in the human NB4 promyelocytic leukemia cell line and primary human hematopoietic stem (CD34+) cells induced to differentiate into neutrophils. Quantitative PCR of Class I and Class IV transcripts showed a positive feedback loop for Class I. Expression of the Class IV transcript was downregulated during hematopoietic cell differentiation. Scatchard analysis showed no differences between the two receptors in high-affinity Kd (∼ 500 nM) for the GCSF-GCSFR. Because Class IV lacks the C-terminal di-leucyl motif that facilitates internalization, we measured internalization rates and found that indeed the Class IV internalized more slowly and less completely. Using an MTT assay to measure proliferation we observed Class IV isoform had lower proliferative capacity at lower GCSF concentrations (0.1 – 2 nM GCSF); however at higher GCSF dose (>100 nM) its proliferative response was greater than Class I. Using western blotting we observed that the Class IV isoform showed weaker signaling via the JAK/STAT and ERK1/2 pathways, but had higher Lyn activity when treated with 100 ng/ml GCSF. To determine effects on differentiation, we made chimeric human growth hormone receptor-GCSFR for transfection into murine 32D cells. 32D cells express low levels of murine GCSFR, thus we made the chimeric receptor. Expression of Class IV Receptor impaired their differentiation (as demonstrated by morphology and Gr-1 expression). We are now developing a mouse model of perturbed hematopoiesis due to dysregulated expression of Class IV GCSFR. Altogether, our studies show that S34F mutation of U2AF1 splicing gene is associated with exon skipping of CSF3R. This would result in expression of a C-terminal truncated GCSFR, similar to that observed in patients with nonsense mutations or alternative splicing. A C-terminal truncated GCSFR causes aberrant hematopoietic cell proliferation, altered post-receptor signaling events, and impaired myeloid differentiation.Our findings and those involving GCSFR E785K in high-risk MDS (Wolfler et al, Blood 105:3731, 2005) strongly suggest that aberrant signaling by alterations in the C-terminus of the GCSFR contributes to the pathogenesis of MDS. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Adiponectin and cardiovascular health: an update

The global epidemic of obesity is accompanied by an increased prevalence of cardiovascular disease (CVD), in particular stroke and heart attack. Dysfunctional adipose tissue links obesity to CVD by secreting a multitude of bioactive lipids and pro-inflammatory factors (adipokines) with detrimental effects on the cardiovascular system. Adiponectin is one of the few adipokines that possesses multiple salutary effects on insulin sensitivity and cardiovascular health. Clinical investigations have identified adiponectin deficiency (hypoadiponectinaemia) as an independent risk factor for CVD. In animals, elevation of plasma adiponectin by either pharmacological or genetic approaches alleviates obesity-induced endothelial dysfunction and hypertension, and also prevents atherosclerosis, myocardial infarction and diabetic cardiomyopathy. Furthermore, many therapeutic benefits of the peroxisome-proliferator activated receptor gamma agonists, the thiazolidinediones, are mediated by induction of adiponectin. Adiponectin protects cardiovascular health through its vasodilator, anti-apoptotic, anti-inflammatory and anti-oxidative activities in both cardiac and vascular cells. This review summarizes recent findings in the understanding of the physiological role and clinical relevance of adiponectin in cardiovascular health, and in the identification of the receptor and postreceptor signalling events that mediate the cardiovascular actions of adiponectin. It also discusses adiponectin-targeted drug discovery strategies for treating obesity, diabetes and CVD. This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-3.

An intervention study in obese mice with astaxanthin, a marine carotenoid – effects on insulin signaling and pro-inflammatory cytokines

Astaxanthin (ASX), a xanthophyll carotenoid from the marine algae Hematococcus pluvialis, has anti-obesity and insulin-sensitivity effects. The specific molecular mechanisms of its actions are not yet established. The present study was designed to investigate the mechanisms underlying the insulin sensitivity effects of ASX in a non-genetic insulin resistant animal model. A group of male Swiss albino mice was divided into two and fed either a starch-based control diet or a high fat-high fructose diet (HFFD). Fifteen days later, mice in each dietary group were divided into two and were treated with either ASX (6 mg kg(-1) per day) in olive oil or olive oil alone. At the end of 60 days, glucose, insulin and pro-inflammatory cytokines in plasma, lipids and oxidative stress markers in skeletal muscle and adipose tissue were assessed. Further, post-receptor insulin signaling events in skeletal muscle were analyzed. Increased body weight, hyperglycemia, hyperinsulinemia and increased plasma levels of tumor necrosis factor-α and interleukin-6 observed in HFFD-fed mice were significantly improved by ASX addition. ASX treatment also reduced lipid levels and oxidative stress in skeletal muscle and adipose tissue. ASX improved insulin signaling by enhancing the autophosphorylation of insulin receptor-β (IR-β), IRS-1 associated PI3-kinase step, phospho-Akt/Akt ratio and GLUT-4 translocation in skeletal muscle. This study demonstrates for the first time that chronic ASX administration improves insulin sensitivity by activating the post-receptor insulin signaling and by reducing oxidative stress, lipid accumulation and proinflammatory cytokines in obese mice.

Endotoxin-Induced Cardiomyopathy and Systemic Inflammation in Mice Is Prevented by Aldose Reductase Inhibition

Sepsis is a systemic inflammatory response syndrome characterized by excessive production of inflammatory cytokines and cardiovascular collapse. Postreceptor signaling events that lead to stress responses and cytokine production are sensitive to redox changes and products of lipid peroxidation. We tested the hypothesis that inflammatory signaling and cytokine generation during sepsis depend on the activity of the enzyme aldose reductase, which catalyzes the reduction of lipid peroxidation-derived aldehydes and their glutathione conjugates. The results of the present study show that pharmacological inhibition of aldose reductase by sorbinil or knockdown of the enzyme by small interfering RNA prevents the activation of nuclear factor-kappaB and the release of tumor necrosis factor-alpha from lipopolysaccharide-stimulated RAW264.7 or H9c2 cells. Increases in serum and cardiac cytokines in response to lipopolysaccharide challenge were suppressed by inhibition of aldose reductase. Treatment with sorbinil blunted the activation of protein kinase C, c-Jun NH2-terminal kinase, and p38, as well as phosphorylation of interleukin receptor-associated kinase, IkappaB-alpha, IkappaB kinase complex-alpha/beta, and phospholipase-gamma1 and -beta1. These changes were associated with decreased myocardial nuclear factor-kappaB and activating protein-1 activity, prostaglandin E2 production, induction of cyclooxygenase 2, and inducible nitric oxide synthase. Sorbinil treatment also induced functional recovery in myocardial fractional shortening in vivo and preserved contractile function of isolated perfused hearts. Inhibition of aldose reductase increased survival in mice injected with lethal doses of lipopolysaccharide. The present demonstration that aldose reductase mediates endotoxin-induced inflammation and cardiomyopathy suggests that inhibition of this enzyme may be useful to attenuate maladaptive host responses and to treat acute cardiovascular dysfunction associated with endotoxic shock.

Possibilities and results in the wide-scale genomic analysis of inflammation

Only recently, available microarray methods have been extended to the investigation of such complex pathophysiological conditions as inflammatory disease. Genome analysis gives the possibility of looking for susceptibility gene loci and their allelic combinations. By the analysis of mRNAs (transcriptome), novel players, functional groups of participants and regulatory pathways involved in the inflammation can be revealed. The possibility of comparing several samples is ideal to increase our knowledge about the kinetics of inflammation. The effects and post-receptor signalling events of individual pro- or anti-inflammatory mediators can also be tested. The use of human post mortem samples could yield otherwise inaccessible information on molecular mechanisms of chronic inflammatory diseases. However, the analysis and interpretation of a huge amount of data, the selection of appropriate experimental systems and time points may hide some pitfalls, which emphasize the need to confirm results with independent methods such as e.g. semi-quantitative Northern, real time PCR, RNA-ase protection assay as well as functional studies.

Activation of nuclear transcription factor-kappa B is associated with the induction of inhibitory kappa B kinase-beta and involves differential activation of protein kinase C and protein tyrosine kinases during fatal murine cerebral malaria

The levels of nuclear transcription factor-kappa B (NF-κB) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to collaborately regulate the NF-κB activation, we also studied the activity of these two kinases in cytosol and membrane fraction. In parallel, the levels of two PKC isoforms (alpha and delta) and tyrosine phosphorylated proteins were monitored to correlate the observed changes in the activity. Our results underscore the involvement of IKK-beta as an essential mediator of NF-κB activation as evinced by the nuclear translocation of p65 and p50 during CM pathology. Additional findings confirm altered activity and levels of PKC and enhanced activation of PTK and tyrosine phosphorylation of proteins during CM pathology. These signaling intricacies involving an interplay between rel family (NF-κB) of transcription factors, PKC and PTK may serve as an important cue in understanding the possible continuation of the post receptor signaling events associated with tumor necrosis factor-alpha induction during FMCM pathology.

Epidermal growth factor induces c-fos and c-jun mRNA via Raf-1/MEK1/ERK-dependent and -independent pathways in bovine luteal cells

Epidermal growth factor (EGF) modulates the actions of gonadotropins in the corpus luteum. The membrane-associated EGF receptors undergo rapid tyrosine phosphorylation and internalization upon ligand binding in ovarian cells, including luteal cells. However, little is known about the post-receptor signaling events induced by EGF that lead to the transcriptional regulation of EGF-responsive genes in the ovary. The present study was designed to examine in bovine luteal cells (1) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling cascade (Raf/MEK/ERK) by EGF; (2) mRNA expression of AP-1 transcription factors, i.e. c-fos and c-jun, in response to EGF; and (3) the role of ERK in EGF-induced expression of c-fos and c-jun mRNA. Raf-1 and B-Raf, but not A-Raf, were activated by EGF (10 ng/ml) and the pharmacological protein kinase C (PKC) activator phorbol myristate acetate (PMA, 20 nM). Activation of Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1) which subsequently activated ERKs. Treatment with EGF-induced the phosphorylation of both ERK2 and ERK1 in a time and concentration dependent manner. Additionally, activated ERK was found in the nucleus of the cells following treatment with EGF (10 ng/ml) and PMA (PMA, 20 nM) for 5 min. Depletion of PKC by chronic PMA treatment (2.5 μM, 24 h) only partially inhibited the stimulatory effects of EGF on Raf-1, ERK2 and ERK1. These data demonstrate that PKC-dependent and independent-mechanisms are involved in EGF activation of the Raf/MEK/ERK signaling cascade in bovine luteal cells. EGF rapidly and transiently stimulated the expression of c-fos and c-jun mRNA in bovine luteal cells. Maximal induction of c-fos and c-jun mRNA by EGF occurred within 30 min of treatment with 10 ng/ml EGF. Treatment with the MEK1 inhibitor PD098059 (50 μM) abolished EGF-induced ERK activation. However, blocking EGF-induced ERK activation by pretreatment with PD098059 only partially attenuated EGF-induced c-fos and c-jun mRNA expression. Thus, additional pathways are implicated in the regulation of c-fos and c-jun mRNA expression by EGF in bovine luteal cells.

NF-kappaB is required for CD38-mediated induction of C(gamma)1 germline transcripts in murine B lymphocytes.

Ligation of CD38 on murine B cells with agonistic anti-CD38 mAb induces B cell proliferation, expression of germline gamma1 transcripts and enhances IL-5 receptor expression. This leads to Ig class switch recombination from the micro to gamma1 heavy chain gene, and high levels of IgM and lgG1 production, particularly in response to anti-CD38 and IL-5 co-stimulation. Although some of the post-receptor signaling events initiated by CD38 ligation have been characterized, signaling pathways involved in CD38-mediated germline gamma1 transcript expression in B cells are poorly understood. Here we show that CD38 ligation of murine splenic B cells activates members of the NF-kappaB/Rel family of proteins including c-Rel, p65 and p50. The activation patterns and kinetics of NF-kappaB-like proteins in CD38-stimulated B cells differ somewhat from those seen in CD40-stimulated B cells. Activation of NF-kappaB-like proteins by CD38 ligation is not observed in splenic B cells from Bruton's tyrosine kinase (Btk)-deficient (Btk(-/-)) mice, with inhibitors of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase also suppressing NF-kappaB activation in CD38-activated B cells. We infer from these results that activation of Btk, PI-3 kinase and PKC play, at least in part, important roles in the induction of NF-kappaB in CD38-stimulated murine B cells. Consistent with a role for NF-kappaB/Rel signaling in CD38-mediated germline gamma1 transcript expression, p50(-/-) B cells show significant impairment of germline gamma1 transcript expression in response to CD38 ligation, whereas the CD40-induced response was not altered. In contrast, c-Rel(-/-) B cells show a severe impairment of germline gamma1 transcript expression in response to CD38 or CD40 ligation. These results indicate an essential role for NF-kappaB proteins in the induction of germline gamma1 transcripts by CD38-ligated murine B cells giving rise to IL-5-induced IgG1 production.

Melatonin and prostate cancer cell proliferation: interplay with castration, epidermal growth factor, and androgen sensitivity.

Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.

Mitochondrial anomalies are associated with the induction of intrinsic cell death proteins-Bcl 2, Bax, cytochrome-c and p53 in mice brain during experimental fatal murine cerebral malaria

The levels of apoptosis associated proteins Bcl 2, Bax, cytochrome-c and p53 was investigated in mice cerebral cortex and cerebellum, using an experimental model of fatal murine cerebral malaria (FMCM). Owing to the activation of events central to mitochondrial dysfunctions, we monitored the structural integrity of mitochondria in cerebral malaria (CM) infected brain tissue by transmission EM (TEM) studies. Western blot analysis revealed the induction of Bcl 2, Bax, cytochrome-c and p53 in both cortex and cerebellum. The TEM studies revealed extensive vacuolation and swelling of mitochondria in infected brain suggestive of a late stage of degeneration. Our results underscore the activation of an intrinsic cell death pathway as evinced by the induction of mitochondria associated apoptotic proteins Bcl 2, Bax and cytochrome-c and further envisages the induction of p53 as a possible continuation of the post receptor signaling events associated with tumor necrosis factor induction following inflammatory responses during CM. These findings may be crucial to mitochondrial dysfunctions underlying the pathology of FMCM.

Differential postreceptor signaling events triggered by excitotoxic stimulation of different ionotropic glutamate receptors in retinal neurons.

The aim of this work was to investigate whether excitotoxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 microM kainate induced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 microM, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D-aspartate receptors. Five hours after stimulation, neuronal retina cells had an apoptotic-like nuclear morphology. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium-dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-kappaB transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 microM kainate or 100 microM glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, although stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that mediate excitotoxic cell death and neuroprotection are stimulus specific.

Essential role of Stat5 for IL-5-dependent IgH switch recombination in mouse B cells.

IL-5 stimulation of CD38-activated murine splenic B cells induces mu-gamma1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent mu-gamma1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced mu-gamma1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a(-/-) and Stat5b(-/-) mice. Expression levels of CD38-induced germline gamma1 transcripts and AID in Stat5a(-/-) and Stat5b(-/-) B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired mu-gamma1 CSR by Stat5b(-/-) B cells, but not by Stat5a(-/-) B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced mu-gamma1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that mu-gamma1 CSR was observed after five or six cell divisions. Stat5a(-/-) and Stat5b(-/-) B cells showed similar cell division cycles, but they did not undergo mu-gamma1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent mu;-gamma1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

A role for endogenous TGFalpha and associated signaling pathways in the differentiation of lens fiber cells.

TGFalpha is hypothesized to be an endogenous regulator of lens fiber terminal differentiation. With immunofluorescence, TGFalpha was localized to differentiating cells in the lens epithelium and superficial fiber cell mass of the adult chicken. A similar pattern of localization was also noted when differentiating epithelial cells were cultured. Immunoneutralization of endogenous TGFalpha inhibited the accumulation of filensin, a unique intermediate filament protein subunit restricted to developing vertebrate lens fibers. ELISA assays quantified the effects of TGFalpha on filensin expression. Surprisingly, inhibition of the TGFalpha receptors' tyrosine kinase activity with nanomolar concentrations of PD153035 increased the accumulation of differentiated characteristics in the presence or absence of ligand. Morphologically, PD153035-treated cells grew as aggregated masses and spread less well onto the substrate. Accompanying these morphologic changes was a complete inhibition of cell division. Post-receptor signaling events were examined with cAMP assays and Western blotting. TGFalpha did not affect cAMP levels while isoproterenol, an additional mediator of lens cell differentiation, caused significant increases in cAMP levels. Activation of ERK2 via dual phosphorylation was noted in response to TGFalpha but not isoproterenol. PD153035 reduced, but did not eliminate, ERK2 phosphorylation in response to TGFalpha. Phosphorylation of the CREB transcription factor was also observed in response to TGFalpha or isoproterenol. These data indicate that endogenous ligands can influence the expression of differentiated characteristics in cultured chick lens cells. A focus of multiple signaling pathways affecting filensin expression is the CREB transcription factor. While increased ERK2 activation may be involved in stimulating cell division, lower levels of persistent ERK2 activation promotes differentiation thus indicating some form of signaling pathway compartmentalization. This could also mean that additional receptors for TGFalpha not inhibited by PD153035 are responsible for promoting differentiation in chick lens cells.

Flt3 Ligand Induces Tyrosine Phosphorylation of Gab1 and Gab2 and Their Association with Shp-2, Grb2, and PI3 Kinase

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.

Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3 have been characterized, the involvement of the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably expressing human Flt3 receptor. Stat5a, but not Stats 1-4, 5b, or 6, was potently activated by Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a required the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of primary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors from marrows of Stat5a(-/-) mice, but did stimulate/costimulate proliferation of these cells from Stat5a(+/+), Stat5b(-/-), and Stat5b(+/+) mice. Thus, Stat5a is essential for at least certain effects of FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

FLT3 Ligand induces functional effects through STAT5a activation and bcl-xl expression as evidenced through biochemical analysis and use of stat 5A −/− mice

Flt3 ligand (FL) is an early acting co-stimulatory cytokine that regulates proliferation, differentiation and survival of hematopoietic stem and progenitor cells. Its receptor, Flt3/Flk2, belongs to the type III receptor tyrosine kinases (RTK) that also include the receptors for M-CSF, Steel factor, and PDGF. Although some post-receptor signaling events and substrate specificities of the Flt3 receptor have been characterized, involvement of the Jak/Stat pathway in Flt3 signaling has not been clearly studied. We found that FL selectively activated Stat5a, without activation of Janus kinases in Baf3/Flt3 cells, a murine IL-3-dependent hematopoietic cell line which stably expresses full-length human Flt3 receptor. FL did not activate Stats 1,2,3,5b, or 6. Activation of Stat5a required the kinase activity of Flt3. We have also examined whether FL upregulates bcl-xL, a STAT5 target gene. Expression of bcl-xL was up-regulated at both the mRNA and protein levels after FL stimulation of Baf3/Flt3 cells. To evaluate physiological relevance of these observations, we assessed responsiveness of myeloid progenitor cells from Stat 5a −/− mice and their littermate controls (+/+) to stimulation by a number of growth factors and co-stimulating molecules, alone and in combination. CFU-GM from Stat 5a −/− and +/+ mice responded similarly to stimulation by various concentrations of GM-CSF, M-CSF, G-CSF, or steel factor (SLF) and to synergism of SLF plus either GM-CSF, M-CSF or G-CSF. In contrast, CFU-GM from control, but not Stat 5a −/−, mice responded to FL and to the synergistic effects of FL with either GM-CSF, M-CSF or G-CSF. In a preliminary experiment, levels of bcl-xl were greatly reduced in Stat 5a −/− compared to +/+ cells. These results demonstrate that Stat5a is essential for the costimulatory effects of FL and suggest that a mechanism for the functional activity of FL is to upregulate expression of bcl-xL through activation of Stat5a.

C5a modulation of interleukin-1 beta-induced interleukin-6 production by human osteoblast-like cells.

Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1beta prior to C5a challenge at optimal concentrations (1.0 microg/ml C5a, 0.1 ng/ml IL-1beta). Cells simultaneously challenged with these concentrations of C5a and IL-1beta produced a 700% increase in IL-6 release relative to cells challenged with IL-1beta alone. Incubation of IL-1beta-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1beta co-stimulation of IL-6. In addition, neither IL-1beta nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1beta on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis.