Stanley W. Halvorsen

Inducers of oxidative stress block ciliary neurotrophic factor activation of Jak/STAT signaling in neurons.

Generation of reactive oxygen species (ROS) with the accumulation of oxidative damage has been implicated in neurodegenerative disease and in the degradation of nervous system function with age. Here we report that ROS inhibit the activity of ciliary neurotrophic factor (CNTF) in nerve cells. Treatment with hydrogen peroxide (H2O2) as a generator of ROS inhibited CNTF‐mediated Jak/STAT signaling in all cultured nerve cells tested, including chick ciliary ganglion neurons, chick neural retina, HMN‐1 motor neuron hybrid cells, and SH‐SY5Y and BE(2)‐C human neuroblastoma cells. H2O2 treatment of non‐neuronal cells, chick skeletal muscle and HepG2 hepatoma cells, did not inhibit Jak/STAT signaling. The H2O2 block of CNTF activity was seen at concentrations as low as 0.1 mm and within 15 min, and was reversible upon removal of H2O2 from the medium. Also, two other mediators of oxidative stress, nitric oxide and rotenone, inhibited CNTF signaling. Treatment of neurons with H2O2 and rotenone also inhibited interferon‐γ‐mediated activation of Jak/STAT1. Depleting the intracellular stores of reduced glutathione by treatment of BE(2)‐C cells with nitrofurantoin inhibited CNTF activity, whereas addition of reduced glutathione protected cells from the effects of H2O2. These results suggest that disruption of neurotrophic factor signaling by mediators of oxidative stress may contribute to the neuronal damage observed in neurodegenerative diseases and significantly affect the utility of CNTF‐like factors as therapeutic agents in preventing nerve cell death.

Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells

Neurons in vivo are exposed to a variety of different growth factors and cytokines. A principal signalling pathway for ciliary neurotrophic factor (CNTF)-like cytokines is the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) system of kinases and transcription factors. In the human cell line (SH-SY5Y), STAT1 and STAT3 activation by CNTF-like cytokines showed tyrosine phosphorylation peaking at 0.5 h and inactivating within 2 h. Tyrosine phosphorylation of the receptor-associated tyrosine kinases Jak1 and Jak2 showed a similar time course of activation and inactivation in response to CNTF. The STAT1 response to the non-CNTF-like cytokine, interferon-gamma (IFN-gamma) did not inactivate. Inactivation to CNTF was not due to a decrease in CNTF receptor subunit gp130 or in levels of Jak1 or Jak2. STAT inactivation was inhibited by the protein kinase blocker H7 and a tyrosine phosphatase blocker, but not by inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) kinase, mTOR-P70/S6 kinase or phosphatidyl inositol-3-kinase (PI-3 kinase). Surprisingly, CNTF caused only a minor increase in levels of suppressors of cytokine signalling, SOCS-1 and SOCS-3. CNTF pretreatment desensitized the cells to the CNTF-like cytokines, leukemia inhibitory factor and oncostatin-M but not to IFN-gamma. These results reveal a complex level of regulation of shared signalling pathways for cytokines that is dependent on both the type of cell and cytokine.

Environmental toxicants inhibit neuronal Jak tyrosine kinase by mitochondrial disruption

Cadmium, mercury and rotenone are environmental pollutants whose neurotoxic mechanisms are not fully understood. We have shown previously that exposure of nerve cells to these agents produces oxidative stress which reversibly blocks growth factor and cytokine-mediated Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signaling. Here we determined a critical role for mitochondrial dysfunction in inhibiting Jak/STAT activity in human BE(2)-C neuroblastoma cells. Exposure of BE(2)-C cells to the heavy metals CdCl(2) and HgCl(2) and to the mitochondrial complex I inhibitor rotenone inhibited interleukin-6, interferon-gamma and ciliary neurotrophic factor-mediated Jak/STAT signaling, reduced Jak1 and Jak2 auto-phosphorylation and induced Jak tyrosine nitration. However, identical exposure of HepG2 hepatoma cells produced no inhibition of these cytokine responses. In contrast, mitochondria in both BE(2)-C and HepG2 cells showed reduced mitochondrial membrane potential and increased superoxide production after exposure to CdCl(2), HgCl(2) and rotenone. Further, in an in vitro Jak auto-phosphorylation assay Jak2 isolated from either BE(2)-C or HepG2 cells was equally inhibited by mitochondria made dysfunctional by treatment with CdCl(2), HgCl(2) and rotenone. Each of these pro-oxidant effects was reversed by the mitochondrial antioxidant alpha-lipoic acid. The actions of cadmium were also blocked by the mitochondrial complex III bypass agent, 2,6-dichloroindophenol. Therefore, in BE(2)-C cells CdCl(2), HgCl(2) and rotenone disrupt mitochondria to increase intracellular ROS, which directly inhibits neuronal Jak tyrosine kinase activity. Non-neuronal cells such as HepG2 cells that are resistant to oxidative stress-mediated inhibition of cytokine signaling possess some as yet unknown mechanism that protects Jak kinases from oxidative insults. Pro-oxidant-induced mitochondrial dysfunction resulting in selective neuronal Jak inhibition provides a potential mechanism for environmental agents to promote neurodegeneration.

Ciliary Neurotrophic Factor Stimulates the Phosphorylation of Two Forms of STAT3 in Chick Ciliary Ganglion Neurons

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that was identified, purified, and cloned based on its neurotrophic activity on cultured chick ciliary ganglion neurons. The molecular mechanisms by which CNTF elicits its effects on these neurons are unknown. We have previously identified functional receptors for CNTF on ciliary ganglion neurons and demonstrated the CNTF-specific tyrosine phosphorylation of an approximately 90-kDa protein. Here we show that CNTF induced the rapid tyrosine phosphorylation and nuclear accumulation of this protein and identify it as an avian form of the transcription factor, STAT3. Identification was confirmed by its recognition with two distinct anti-STAT3 antibodies and the lack of binding to antibodies against STAT1, -2, -4, -5, or -6. The phosphorylation was stable for up to 2 h but required the continued presence of CNTF. CNTF also induced the tyrosine phosphorylation of a similar protein in cultured chick dorsal root ganglion and retinal neurons. In addition, we identify a second, 100-kDa form of STAT3 that appears in response to CNTF. Unlike previous reports, utilizing mammalian cell lines that detected a slower migrating form of STAT3 resulting from H7-sensitive protein phosphorylation, H7 did not prevent the appearance of the 100-kDa form in ciliary neurons. Thus, the 100-kDa avian protein may represent a novel form of CNTF-inducible STAT3.

Channel activators regulate ATP-sensitive potassium channel (KIR6.1) expression in chick cardiomyocytes

ATP‐sensitive potassium channels (KATP) are widely expressed and yet little is known about the mechanisms regulating their expression. Here we report that expression of chick heart Kir6.1 is regulated by channel activators. Activation of KATP with either ATP depletion or pinacidil, up‐regulated Kir6.1 mRNA 1.8‐ to 2.4‐fold in cultured ventricular myocytes as measured by competitive PCR. Pinacidil treatment also increased Kir6.1 protein as detected using an antibody to Kir6.1. Glibenclamide, a KATP inhibitor, completely blocked the pinacidil‐induced increase in Kir6.1 levels. It appears that Kir6.1 is up‐regulated by an unknown signal transduction pathway initiated by KATP opening.

Opposing regulation of ciliary neurotrophic factor receptors on neuroblastoma cells by distinct differentiating agents

We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.

Identification of functional receptors for ciliary neurotrophic factor on chick ciliary ganglion neurons.

Ciliary neurotrophic factor and an avian homolog, growth promoting activity, are members of the cytokine/neurokine family of trophic factors and have been proposed to function as survival and developmental factors for ciliary ganglion neurons in vivo. Here we identify for the first time functional receptors for ciliary neurotrophic factor and growth promoting activity on cultured ciliary ganglion neurons. [(125)I]Rat ciliary neurotrophic factor binding studies indicate that rat ciliary neurotrophic factor and growth promoting activity bind to these receptors with a single affinity, while human ciliary neurotrophic factor recognizes both a high- and low-affinity site. Comparison of the relative potency of human ciliary neurotrophic factor and avian growth promoting activity in biological assays indicates that growth promoting activity is three to five times more active in promoting survival and in regulating acetylcholine receptors. The binding of ciliary neurotrophic factor is specific, sensitive to phosphatidylinositol-specific phospholipase C and partially inhibited by leukemia inhibitory factor, but not inhibited by other members of the human neurokine family, including interleukin-6, interleukin-22 and oncostatin M. Cross-linking of [(125)I]rat ciliary neurotrophic factor to ciliary neurons results in the specific labeling of three proteins with estimated molecular masses of 153,000, 81,000 and 72,000. Only the 81,000 molecular weight component is released from the cells after treatment with phosphatidylinositol-specific phospholipase C, suggesting a membrane attachment via a glycosylphosphatidylinositol linkage. Stimulation with ciliary neurotrophic factor or growth promoting activity, but not by other neurokines, results in the rapid tyrosine phosphorylation of a 90,000 molecular weight protein that is inhibited by pretreatment with phosphatidylinositol-specific phospholipase C. In conclusion, we report here the pharmacological and functional properties of ciliary neurotrophic factor receptors on embryonic ciliary ganglion neurons. These results provide the means for elaborating the molecular mechanisms of ciliary neurotrophic factor action and understanding its physiological role in a defined neuronal population.

Induction of an interferon‐γ Stat3 response in nerve cells by pre‐treatment with gp130 cytokines

Many cytokines mediate their effects through Jak/STAT signaling pathways providing many opportunities for cross‐talk between different cytokines. We examined the interaction between two cytokine families, gp130‐related cytokines and interferon‐γ (IFN‐γ), which are coexpressed in the nervous system during acute trauma and pathological conditions. Typical nerve cells show an IFN‐γ response that is restricted to activating STAT1, with minor activation of STAT3. IFN‐γ elicited a pronounced STAT3 response in cells pre‐treated for 5–7 h with ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or interleukin‐6. CNTF or interleukin‐6 induced an IFN‐γ STAT3 response in a variety of cells including SH‐SY5Y human neuroblastoma, HMN‐1 murine motor neuron hybrid cells, rat sympathetic neurons and human hepatoma HepG2 cells. The enhancement was measured as an increase in tyrosine phosphorylated STAT3, in STAT3‐DNA binding and in STAT‐luciferase reporter gene activity. The enhanced STAT3 response was not due to an increase in overall STAT3 levels but was dependent upon ongoing protein synthesis. The induction by CNTF was inhibited by the protein kinase C inhibitor, BIM, and the MAPK‐kinase inhibitor, U0126. Further, H‐35 hepatoma cells expressing gp130 receptor chimeras lacking either the SHP‐2 docking site or the Box 3 STAT binding sites failed to enhance the IFN‐γ STAT3 response. These results provide evidence for an interaction between gp130 and IFN‐γ cytokines that can significantly alter the final cellular response to IFN‐γ.

Retinoic acid up-regulates ciliary neurotrophic factor receptors in cultured chick neurons and cardiomyocytes

Retinoic acid is an important developmental factor in the heart and nervous system and regulates the expression of trophic factor receptors in neural cell lines. Here we show the effects of retinoic acid on cytokine responsiveness in embryonic chick neurons and myocytes. Treatment of cultured cardiomyocytes and retinal and ciliary ganglion neurons with retinoic acid resulted in increased expression of receptors for the neuropoietic cytokine, CNTF. All-trans-retinoic acid induced as much as a 3-fold increase in CNTF receptor alpha subunit mRNA in a time and concentration dependent manner and resulted in an enhanced CNTF-induced tyrosine phosphorylation of the transcription factor, STAT3. These results indicate that neurons and myocytes expressing CNTF receptors are responsive to retinoic acid and suggest that retinoids may regulate cell sensitivity to cytokines during development.

Initiation and maintenance of CNTF-Jak/STAT signaling in neurons is blocked by protein tyrosine phosphatase inhibitors

Cytokines, including interferon-gamma and ciliary neurotrophic factor (CNTF), act in common through tyrosine kinase-based Jak/STAT signaling pathways. We found that activation of the Jak/STAT pathway by both interferon-gamma and CNTF in nerve cells was rapidly terminated by tyrosine phosphatase inhibitors. Exposure of human neuroblastoma cells, BE(2)-C, first to tyrosine phosphatase inhibitors (either phenylarsine oxide or PTP inhibitor-2) prevented Jak1, STAT1 and STAT3 activation elicited subsequently by either CNTF or interferon-gamma. In contrast, exposure of these cells to phosphatase inhibitors after initial stimulation by CNTF or interferon-gamma prevented the normal time-dependent decrease of total cellular phosphotyrosine-STAT levels as expected, while excluding already formed phosphotyrosine-STAT from the nucleus. Thus, treatment of nerve cells with a tyrosine phosphatase inhibitor blocked nuclear signal transduction. A similar inhibition of CNTF-Jak/STAT signaling was observed following tyrosine phosphatase inhibition in SH-SY5Y human neuroblastoma cells, HMN-1 mouse motor neuron-neuroblastoma hybrid cells, HepG2 human hepatoma cells and embryonic chick ciliary ganglion and retinal neurons. Expression of dominant-negative forms of the tyrosine phosphatases, SHP-1 and/or SHP-2, in BE(2)-C cells had no effect on CNTF activation of STAT or on the ability of phosphatase inhibitors to block signaling. Further, results from H-35 cells expressing gp130 receptor subunits lacking functional SHP-2 binding sites revealed normal cytokine activation of Jak and STAT that was inhibited by phosphatase inhibitors. These findings suggest a critical control for regulating the initiation of Jak/STAT signaling requiring tyrosine phosphatase activity.