Chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells in a PKC-independent manner

Abstract

Background: Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells.

Methods: In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line.

Results: We found that 10μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca²⁺ levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca²⁺ chelator BAPTA-AM.

Conclusions: This is the first report showing that chelerythrine promotes Ca²⁺-dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound.

General significance: Chelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.

Keywords: Cleaved caspase-3; ERK1/2; PKA; Spectrin breakdown products; Striatal-enriched protein tyrosine phosphatase.