Bryostatins 1 and 3 inhibit TRPM8 and modify TRPM8- and TRPV1-mediated lung epithelial cell responses to a proinflammatory stimulus via protein kinase C.

Abstract

Bryostatin 1 is a protein kinase C (PKC α, β, δ) activator with anti-inflammatory effects. We hypothesized that bryostatins 1 and 3 could modulate transient receptor potential (TRP) channels via PKC and alter TRP-mediated proinflammatory signaling in lung epithelial cells challenged with a proinflammatory stimulus, coal fly ash (CFA). Bryostatins 1 and 3 inhibited icilin-induced calcium flux in HEK-293 cells overexpressing full-length human transient receptor potential melastatin-8 (TRPM8) but did not inhibit activation by menthol or the activities of human transient receptor potential ankyrin 1, transient receptor potential vanilloid 1 (TRPV1), TRPV3, or TRPV4; mouse and rat TRPM8 were less sensitive to inhibition. TRPM8 inhibition was transient (<24 hours), PKC-dependent, and involved differential phosphorylation of amino acids T17, S27, S850, and S1040. CFA particles stimulate interleukin-8 (IL8) and C-X-C motif chemokine ligand 1 (CXCL1) expression by human bronchial epithelial cells via activation of truncated TRPM8 (TRPM8-Δ801) and TRPV1. However, bryostatins 1 and 3 altered IL8 and CXCL1 mRNA expression with and without CFA treatment. At 4 hours, the bryostatins also suppressed TRPM8 mRNA and induced TRPV1 mRNA, which reversed at 24 hours. These effects were reversed by pharmacological inhibition of PKC isoforms (α, ζ, ε, or η) but not δ, implying a network comprised of presumably PKCα, TRPM8-Δ801, and TRPV1 that regulates IL8 and CXCL1 expression by airway epithelial cells. Finally, an unexpected interaction between TRPV1 and TRPM8, but not TRPM8-Δ801, was also identified. Specifically, the coexpression of TRPM8 and TRPV1 reduced TRPM8 expression and activity, which was reversed by TRPV1 inhibition, revealing novel mechanisms by which bryostatins and PKC affect TRP channel signaling in lung epithelial and potentially other cell types. SIGNIFICANCE STATEMENT: Bryostatins 1 and 3 selectively and transiently inhibit human TRPM8 activity via protein kinase C-dependent phosphorylation and temporally modify the expression and induction of interleukin-8 and C-X-C motif chemokine ligand 1 in lung epithelial cells by regulating TRPV1 and TRPM8 expression. This regulatory nexus may have therapeutic potential for treating airway inflammation.