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. 2009 Jan;108(1):246-59.
doi: 10.1111/j.1471-4159.2008.05759.x. Epub 2008 Nov 21.

CNTF-evoked Activation of JAK and ERK Mediates the Functional Expression of T-type Ca2+ Channels in Chicken Nodose Neurons

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Free PMC article

CNTF-evoked Activation of JAK and ERK Mediates the Functional Expression of T-type Ca2+ Channels in Chicken Nodose Neurons

Thomas Trimarchi et al. J Neurochem. .
Free PMC article

Abstract

Culture of chicken nodose neurons with CNTF but not BDNF causes a significant increase in T-type Ca(2+) channel expression. CNTF-induced channel expression requires 12 h stimulation to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. In this study, we have investigated the biochemical mechanism responsible for the CNTF-dependent stimulation of T-type channel expression in nodose neurons. Stimulation of nodose neurons with CNTF evoked a considerable increase in signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation. CNTF-evoked ERK phosphorylation was transient whereas BDNF-evoked activation of ERK was sustained. Pre-treatment of nodose neurons with the Janus tyrosine kinase (JAK) inhibitor P6 blocked STAT3 and ERK phosphorylation, whereas the ERK inhibitor U0126 prevented ERK activation but not STAT3 phosphorylation. Both P6 and U0126 inhibited the stimulatory effect of CNTF on T-type channel expression. Inhibition of STAT3 activation by the selective blocker stattic has no effect on ERK phosphorylation and T-type channel expression. These results indicate that CNTF-evoked stimulation of T-type Ca(2+) channel expression in chicken nodose neurons requires JAK-dependent ERK signaling. A cardiac tissue extract derived from E20 chicken heart was also effective in promoting T-type Ca(2+) channel expression and STAT3 and ERK phosphorylation. The ability of the heart extract to stimulate JAK/STAT and ERK activation was developmentally regulated. These findings provide further support to the idea that CNTF or a CNTF-like factor mediates normal expression of T-type channels.

Figures

Figure 1
Figure 1
Effect of BDNF, chick CNTF and heart extract on T-type Ca2+ channel expression in vitro. A) Representative traces of inward Ca2+ currents of E7 nodose neurons cultured for 24 hr with BDNF (50 ng/mL) or chCNTF (50 ng/mL). T-type Ca2+ currents were generated by a 200 ms depolarizing pulses to −20 mV from a holding potential of −100 mV (filled arrows). HVA Ca2+ currents were generated by a 200 ms depolarizing pulse to +20 mV from a holding potential of −100 mV (empty arrows). Stimulation protocol for both T-type and HVA Ca2+ currents is shown in bottom trace. B) Mean T-type Ca2+ current densities after 24 hr treatment with BDNF, chCNTF and heart extract as compared with acutely isolated E7 nodose neurons (control). Current densities were obtained by dividing current amplitude by cell capacitance. Note little differences in T-type Ca2+ current densities between acutely isolated and BDNF-treated neurons. Culture of nodose neurons with chCNTF or heart extract evokes a significant increase in T-type Ca2+ current densities (* denotes p ≤ 0.05 vs. BDNF). C) Culture of E7 nodose neurons with BDNF, chick CNTF or heart extract does not alter mean HVA Ca2+ current densities (ns=not significant).
Figure 2
Figure 2
Expression pattern of CNTFRα, LIFRβ and gp130 transcripts in chick nodose neurons. A, B, C) Relative expression of CNTFRα, LIFRβ and gp130 transcripts in chick nodose neurons as determined by real time PCR. Plots of the relative expression of CNTFRα, LIFRβ and gp130 mRNA as a function of age. * and ** denote p ≤ 0.05 vs. E7 and E20, respectively.
Figure 3
Figure 3
Time course of STAT3 and ERK activation in nodose sensory neurons following stimulation with chCNTF or BDNF. A, B) Stimulation of nodose cell cultures with chCNTF generates a considerable increase in STAT3 and ERK phosphorylation. C) Phosphorylation pattern of STAT3 as determined by the intensity ratio of pSTAT3 to total STAT3. Stimulation of nodose neurons with chCNTF for 30 min causes a significant increase in the pSTAT3/STAT3 ratio. D) Phosphorylation pattern of ERK as determined by the intensity ratio of pERK to total ERK. Note that stimulation with chCNTF for 30 min causes a significant increase in the pERK/ERK ratio. The pERK/ERK intensity ratio decreases significantly after 3 hr stimulation with CNTF (n=8). E, F) Stimulation of nodose cell cultures with BDNF results in a significant increase in ERK activation without evoking STAT3 activation. G) The intensity ratio of pSTAT3 to total STAT3 was very low following stimulation of nodose neurons with BDNF. Notice that the scale of the Y-axis in figures C and G are the same in order to visualize differences in the pattern of STAT3 activation evoked by chCNTF and BDNF. H) As determined by the pERK/total ERK intensity ratio, BDNF stimulation of nodose neurons causes a significant increase in ERK phosphorylation, that was maintained for up to 3 hr (n=7). Cell cultures of nodose neurons were treated with chCNTF (50 ng/mL) or BDNF (50 ng/mL) for various lengths of time (5 min, 30 min and 3 hr). Cell lysates were collected and subjected to immunoblot analysis using a two-color western blot detection with the Odyssey infrared imaging system. * denotes p ≤ 0.05 vs. control (no treatment), ** denotes p ≤ 0.05 vs. CNTF treatment for 30 min.
Figure 4
Figure 4
Effect of the JAK inhibitor P6 and the ERK inhibitor U0126 on STAT3 and ERK phosphorylation in nodose neurons. A, B) Chick CNTF evoked activation of STAT3 and ERK is blocked by the inhibitor of JAK kinases P6 (10 μM). C, D) The ERK inhibitor U0126 (50 μM) only blocks ERK phosphorylation but does not affect STAT3 activation. E, F) Effect of P6 and U0126 on the pSTAT3/STAT3 and pERK/total ERK intensity ratio. Stimulation of nodose neurons with chCNTF caused a significant increase in the pSTAT3/total STAT3 ratio. Notice that only P6 but not U0126 caused a significant reduction in the pSTAT3/STAT3 intensity ratio. Stimulation of nodose neurons with chCNTF also caused a 3-fold increase in the pERK/total ERK ratio. Treatment with either P6 or U0126 inhibits the stimulatory effect of chCNTF on ERK phosphorylation (n= 3–5). In these experiments, nodose neurons were isolated at E7 and pre-treated with P6 (or U0126) for 1 hr prior exposure to chCNTF for 30 min. * denotes p ≤ 0.05 vs. control (no treatment), ** denotes p ≤ 0.05 vs. CNTF treatment for 30 min.
Figure 5
Figure 5
Effect of JAK/STAT and ERK signaling inhibitors on the CNTF-evoked stimulation of T-type Ca2+ channel expression. A) The JAK inhibitor P6 blocked the stimulatory effect of chCNTF on T-type Ca2+ channel expression. B) Inhibition of ERK activation with U0126 blocked T-type Ca2+ channel expression evoked by CNTF. In these experiments, nodose neurons were isolated at E7 and maintained in culture for 12 hr in the presence of CNTF (50 ng/mL). The culture medium was also supplemented with BDNF (50 ng/mL) to promote neuronal survival. Controls represent BDNF-treated cultures. Cultures were pre-treated with P6 or U0126 for 1 hr prior stimulation with CNTF. * denotes p ≤ 0.05 vs. control; ** denotes p ≤ 0.05 vs. chCNTF.
Figure 6
Figure 6
Effect of the STAT3 inhibitor stattic on the CNTF-evoked stimulation of T-type Ca2+ channel expression and activation of JAK/STAT3 and ERK. A, B) At 2 μM stattic caused a significant reduction of STAT3 phosphorylation without affecting ERK activation. C, D) At higher concentrations (20 μM), stattic eliminated the CNTF-induced STAT3 phosphorylation without any noticeable effect on ERK activation. Notice that when applied alone, stattic does not cause any noticeable effect on the basal levels of STAT3 and ERK phosphorylation. E–F) Effect of stattic on the pSTAT3/STAT3 and pERK/total ERK intensity ratios. Notice that only pSTAT3/STAT3 but not pERK/total ERK intensity ratio is affected by stattic. Nodose neurons were isolated at E7 and pre-treated with stattic for 1 hr prior exposure to chCNTF for 30 min. G) Inhibition of STAT3 phosphorylation by stattic did not alter T-type Ca2+ channel expression evoked by chCNTF (* denotes p ≤ 0.05 vs. control). In these experiments, nodose neurons were isolated at E7 and maintained in culture for 12 hr in the presence of CNTF (50 ng/mL). The culture medium was also supplemented with BDNF (50 ng/mL) to promote neuronal survival. Controls were exposed to BDNF alone. Cultures were pre-treated with stattic for 1 hr prior stimulation with chCNTF. * denotes p ≤ 0.05 vs. chCNTF (E) or control (G), whereas ns indicates that no significant differences were detected between groups.
Figure 7
Figure 7
Time course of STAT3 and ERK activation in BE(2)-C cells following stimulation with chCNTF (A, B) or heart extract (C, D). E, F) Stimulation of nodose cell cultures with heart extract for 30 min also generated a considerable increase in STAT3 and ERK phosphorylation. Cell cultures of BE(2)-C cells or nodose neurons were treated with CNTF (50 ng/mL) for various lengths of time. Cell lysates were collected and subjected to immunoblot analysis using a two-color western blot detection with the Odyssey infrared imaging system.
Figure 8
Figure 8
The ability of the heart extract to evoke STAT3 and ERK phosphorylation is developmentally regulated. A, B) The E7 heart extract was less effective in eliciting STAT3 phosphorylation when compared with nodose neurons stimulated with an E20 heart extract. C, D) The stimulatory effect of the E7 heart extract on ERK phosphorylation was considerably lower than that generated by the E20 heart extract. Cell cultures of nodose neurons were treated with an E7 or E20 heart extract (at 200 μg/mL) for 30 min. Cell lysates were collected and subjected to immunoblot analysis of STAT3 and ERK phosphorylation. * denotes p ≤ 0.05 vs. non-stimulated samples.

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