Cannabinoid 1 receptor and interleukin-6 receptor together induce integration of protein kinase and transcription factor signaling to trigger neurite outgrowth.
ABSTRACT Activation of the G(o/i)-coupled cannabinoid 1 receptor (CB1R) has been shown to induce neurite outgrowth in Neuro2A cells through activation of Src kinase and STAT3 transcription factor. Signaling by the interleukin 6 receptor (IL-6R) also activates STAT3 through Jak kinase. We studied if signals from the two pathways could be integrated in a synergistic manner to trigger neurite outgrowth in Neuro2A cells. At low concentrations, when agonist at either receptor by itself has no effect, we found that CB1R and IL-6R stimulation together induced synergistic neurite outgrowth. Signal integration requires activation of transcription factors by Src, Jak, and mitogen-activated protein kinases. Mitogen-activated protein kinase can be activated by both receptors and shows enhanced early activation in the presence of both ligands. CREB and STAT3 transcription factors are required for synergy and show enhanced DNA-binding activity when both receptors are activated. STAT3 plays a critical role in integration of the signals downstream of the two receptors. When both pathways are activated, STAT3 phosphorylation is sustained for 6 h. This prolonged activation of STAT3 requires deactivation of SHP2 phosphatase. Reduction of SHP2 levels by RNA interference results in greater synergy in neurite outgrowth. Simultaneous knockdown of both SHP2 and STAT3 blocks the synergistic triggering of neurite outgrowth, indicating that STAT3 is downstream of SHP2. CB1R and IL-6R co-stimulation enhanced the differentiation of rat cortical neuron primary cultures. These results provide a mechanism where multiple protein kinases and transcription factors interact to integrate signals from G protein-coupled and cytokine receptor to evoke neurite outgrowth in Neuro2A cells.
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ABSTRACT: Neurotrophins are traditionally known for their roles in neuronal development, function and survival. More recent data has highlighted the importance of neurotrophin signalling in adult signalling contexts, including the regulation of synaptic transmission. In addition, neurotrophin levels are increased in inflammatory and neuropathic pain leading to sensitization to painful stimuli. Endocannabinoid (eCB) signalling was initially studied in the context of synaptic transmission and pain alleviation whilst recently gaining attention due to its involvement in the development of the nervous system. Similar to neurotrophins, eCB levels also rise during pain perception but result in diminished pain sensations. The overlap of cellular functions between neurotrophins and eCB signalling leads to the hypothesis that these signalling systems are positioned to regulate each other and narrow the multitude of actions that both systems can promote to the specific need of the cell. Therefore, in this review, we examine to what extent the involvement of these two signalling systems is co-ordinated as opposed to being coincidental, and causal to neuronal circuit modifications in pain. Available data point to numerous direct molecular interactions between the neurotrophin and eCB signalling systems in developmental and adult contexts, including receptor-level interplay, transcriptional control and synergistic regulation of downstream signalling cascades. Although experimental observations specifically in pain circuits are limited, the universality of downstream signalling systems from both neurotrophin and endocannabinoid receptors suggest an interdependent relationship between these two diverse signalling systems.European Journal of Neuroscience 02/2014; 39(3):334-43. DOI:10.1111/ejn.12431 · 3.67 Impact Factor
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ABSTRACT: Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension. © 2014 Wiley Periodicals, Inc.Journal of Neuroscience Research 02/2015; 93(2). DOI:10.1002/jnr.23482 · 2.73 Impact Factor
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ABSTRACT: A luminex-based screen of cytokine expression in dorsal root ganglia (DRG) and nerve of type 1 diabetic rodents revealed interleukin-1 (IL-1alpha) and IL-1beta to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1alpha and IL-1beta expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy. In addition, we determined if these cytokines could optimize mitochondrial bioenergetics since mitochondrial dysfunction is a key etiological factor in diabetic neuropathy. Cytokines IL-1alpha and IL-1beta were reduced 2-fold (p<0.05) in DRG and/or nerve of 2 and 5 month streptozotocin (STZ)-diabetic rats. IL-2 and IL-10 were unchanged. IL-1alpha and IL-1beta induced similar 2 to 3-fold increases in neurite outgrowth in cultures derived from control or diabetic rats (p<0.05). STAT3 phosphorylation on Tyr705 or Ser727 was depressed in DRG from STZ-diabetic mice and treatment of cultures derived from STZ-diabetic rats with IL-1beta for 30 min raised phosphorylation of STAT3 on Tyr705 and Ser727 by 1.5 to 2-fold (p<0.05). shRNA-based or AG490 inhibition of STAT3 activity or shRNA blockade of endogenous IL-1beta expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1beta and maximal oxygen consumption rate and spare respiratory capacity, both key measures of bioenergetic fidelity that were depressed in diabetic compared with control neurons, were enhanced 2-fold. This effect was blocked by AG490. Endogenous synthesis of IL-1beta is diminished in nerve tissue in type 1 diabetes and we propose this defect triggers reduced STAT3 signaling and mitochondrial function leading to sup-optimal axonal regeneration and plasticity.Molecular Brain 10/2013; 6(1):45. DOI:10.1186/1756-6606-6-45 · 4.35 Impact Factor