Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.
ABSTRACT: Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.
Project description:Transient receptor potential canonical (TRPC) proteins, a group of Ca2' permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC) and mediate store-operated calcium entry (SOCE) in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC).Real-time quantitative PCRwas performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot.Among the seven family members of TRPC so far identified (TRPC1-7), we detected the expression ofTRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1 approximately equal TRPC6 > TRPC3 > TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue.Out of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.
Project description:Plasma epinephrine and heart rate are elevated in metabolic syndrome, suggesting enhanced catecholamine secretion from the adrenal medulla. Canonical transient receptor potential (TRPC) channels are implicated in mediating hormone-induced Ca(2+) influx and catecholamine secretion in adrenomedullary chromaffin cells. We studied the pattern of TRPC expression in the pig adrenal medulla and investigated whether adrenal TRPC expression is altered in prediabetic metabolic syndrome Ossabaw miniature pigs. We used a combination of molecular biological, biochemical, and fluorescence imaging techniques. We determined the sequence of pig TRPC1 and TRPC3-7 channels. We found that the pig adrenal medulla expressed predominantly TRPC1, TRPC5, and TRPC6 transcripts. The expression level of these TRPCs was significantly elevated in the adrenal medulla from pigs with metabolic syndrome. Interestingly, aldosterone, which is endogenously secreted in the adjacent adrenal cortex, increased TRPC1, TRPC5, and TRPC6 expression in adrenal chromaffin cells isolated from metabolic syndrome but not control pigs. Spironolactone, a blocker of mineralocorticoid receptors, inhibited the aldosterone effect. Dexamethasone also increased TRPC5 expression in metabolic syndrome chromaffin cells. The amplitude of hormone-induced divalent cation influx correlated with the level of TRPC expression in adrenal chromaffin cells. Orai1/Stim1 protein expression was not significantly altered in the metabolic syndrome adrenal medulla when compared with the control. We propose that in metabolic syndrome, abnormally elevated adrenal TRPC expression may underlie increased plasma epinephrine and heart rate. The excess of plasma catecholamines and increased heart rate are risk factors for cardiovascular disease. Thus, TRPCs are potential therapeutic targets in the fight against cardiovascular disease.
Project description:The expression of Nex1 peaks during brain development when neurite outgrowth and synaptogenesis are highly active. We previously showed that Nex1 is a critical effector of the nerve growth factor (NGF) pathway and its overexpression results in spontaneous neuritogenesis. Furthermore, the PC12-Nex1 cells exhibit accelerated neurite extension upon NGF exposure, and have the capacity to regenerate neurites in the absence of NGF. In this study, we identify the repertoire of genes targeted by Nex1 to unravel the molecular mechanisms by which Nex1 promotes differentiation and regeneration. Our transcriptional analysis reveals that Nex1 modulates a wide spectrum of genes with diverse functions, many of them being key downstream regulators of the NGF pathway, and critical to neuritogenesis, such as microtubules, microtubule-associated proteins (MAPs) and intermediate filaments. We also provide the first evidence that a basic helix-loop-helix (bHLH) protein stimulates the expression of the cyclin-dependent kinase (CDK) inhibitors belonging to the INK4 family, which plays a role in promoting cell-cycle arrest. Finally, we show a dramatic synergistic effect between Nex1 and cAMP, resulting in an impressive regeneration of an elaborate and dense neurite network. Thus, Nex1 has endowed the PC12-Nex1 cells with a distinct combination of gene products that takes part in the complex regulation of neuritogenesis and regeneration.
Project description:Store-operated Ca(2+) entry (SOCE) channels are the main pathway of Ca(2+) entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5(+) NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5(+) NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5(+) NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5(+) NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5(+) NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs.
Project description:Stromal interacting molecule 1 (STIM1) is a Ca(2+) sensor that conveys the Ca(2+) load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La(3+), suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.
Project description:X-linked Inhibitor of apoptosis protein (XIAP) has been classically identified as a cell death regulator. Here, we demonstrate a novel function of XIAP as a regulator of neurite outgrowth in neuronal cells. In PC12 cells, XIAP overexpression prevents NGF-induced neuronal differentiation, whereas NGF treatment induces a reduction of endogenous XIAP levels concomitant with the induction of neuronal differentiation. Accordingly, downregulation of endogenous XIAP protein levels strongly increases neurite outgrowth in PC12 cells as well as axonal and dendritic length in primary cortical neurons. The effects of XIAP are mediated by the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERKs) pathway since blocking this pathway completely prevents the neuritogenesis mediated by XIAP downregulation. In addition, we found that XIAP binds to cRaf and Trk receptors. Our results demonstrate that XIAP plays a new role as a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons.
Project description:Hericium erinaceus is a culinary-medicinal mushroom used traditionally in Eastern Asia to improve memory. In this work, we investigated the neuroprotective and neuritogenic effects of the secondary metabolites isolated from the MeOH extract of cultured mycelium of H. erinaceus and the primary mechanisms involved. One new dihydropyridine compound (6) and one new natural product (2) together with five known compounds (1,3-5,7) were obtained and their structures were elucidated by spectroscopic analysis, including 2D NMR and HRMS. The cell-based screening for bioactivity showed that 4-chloro-3,5-dimethoxybenzoic methyl ester (1) and a cyathane diterpenoid, erincine A (3), not only potentiated NGF-induced neurite outgrowth but also protected neuronally-differentiated cells against deprivation of NGF in PC12 pheochromocytoma cells. Additionally, compound 3 induced neuritogenesis in primary rat cortex neurons. Furthermore, our results revealed that TrkA-mediated and Erk1/2-dependant pathways could be involved in 1 and 3-promoted NGF-induced neurite outgrowth in PC12 cells.
Project description:We provide the first characterization of a novel signaling adapter, Nesca, in neurotrophic signal transduction. Nesca contains a RUN domain, a WW domain, a leucine zipper, a carboxyl-terminal SH3 domain, and several proline-rich regions. Nesca is highly expressed in the brain, is serine phosphorylated, and mobilizes from the cytoplasm to the nuclear membrane in response to neurotrophin, but not epidermal growth factor, stimulation in a MEK-dependent process. Overexpression studies in PC12 cells indicate that Nesca facilitates neurotrophin-dependent neurite outgrowth at nonsaturating doses of nerve growth factor (NGF). Similarly, short interfering RNA studies significantly reduce NGF-dependent neuritogenesis in PC12 cells. Mutational analyses demonstrate that the RUN domain is an important structural determinant for the nuclear translocation of Nesca and that the nuclear redistribution of Nesca is essential to its neurite outgrowth-promoting properties. Collectively, these works provide the first functional characterization of Nesca in the context of neurotrophin signaling and suggest that Nesca serves a novel, nuclear-dependent role in neurotrophin-dependent neurite outgrowth.
Project description:Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.
Project description:Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.