Clozapine induces chloride channel-4 expression through PKA activation and modulates CDK5 expression in SH-SY5Y and U87 cells

Prog Neuropsychopharmacol Biol Psychiatry. 2015 Jan 2;56:168-73. doi: 10.1016/j.pnpbp.2014.09.002. Epub 2014 Sep 20.


Objectives: Second-generation antipsychotic drugs, such as clozapine, were reported to enhance neurite outgrowth by nerve growth factor in PC12 cells. The authors previously showed that chloride channel 4 (CLC-4) is responsible for nerve growth factor-induced neurite outgrowth in neuronal cells. In this study, we examined whether clozapine induces CLC-4 in neuroblastoma and glioma cells.

Methods: The effect of clozapine on CLC-4 expression was examined in neuroblastoma (SH-SY5Y) and glioma (U87) cells. To investigate the signaling pathway responsible for clozapine-induced CLC-4 expression, the phosphorylation of cAMP response element-binding protein (CREB), which binds CRE in the promoter of the human CLC-4 gene, was examined. To identify the target of clozapine induced CLC-4, CLC-4 siRNA was introduced to neuroblastoma and glioma cells for functional knockdown.

Results: We observed that clozapine increased CLC-4 expression in both SH-SY5Y and U87 cells. Clozapine induced CREB phosphorylation, but in the presence of inhibitor of protein kinase A (an upstream kinase of CREB) clozapine-induced CLC-4 expression was suppressed. Finally, we found that CLC-4 knockdown suppressed clozapine-induced cyclin-dependent kinase 5 (CDK5) expression in SH-SY5Y and U-87 cells suggesting CDK5 as potential molecular target of clozapine induced CLC-4 expression.

Conclusions: The results of the present study suggest that clozapine's therapeutic effect may include the induction of CLC-4 which is dependent on CREB activation via PKA. We also found that functional knockdown of CLC-4 resulted in reduction of CDK5 expression, which may also be implicated in clozapine's therapeutic effect.

Keywords: Chloride channel-4; Clozapine; Cyclin-dependent kinase 5; SH-SY5Y; U87.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Antipsychotic Agents / pharmacology*
  • CREB-Binding Protein / metabolism
  • Cell Line, Tumor
  • Clozapine / pharmacology*
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cyclin-Dependent Kinase 5 / genetics
  • Cyclin-Dependent Kinase 5 / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Gene Expression Regulation, Neoplastic / drug effects*
  • Glioblastoma / pathology
  • Humans
  • Nerve Growth Factor / pharmacology
  • Neuroblastoma / pathology
  • RNA, Small Interfering / pharmacology


  • Antipsychotic Agents
  • RNA, Small Interfering
  • Nerve Growth Factor
  • CREB-Binding Protein
  • Cyclin-Dependent Kinase 5
  • Cyclic AMP-Dependent Protein Kinases
  • CDK5 protein, human
  • Clozapine