A steroid alkaloid derivative 02F04 upregulates thymic stromal lymphopoietin expression slowly and continuously through a novel Gq/11-ROCK-ERK1/2 signaling pathway in mouse keratinocytes

Abstract

Thymic stromal lymphopoietin (TSLP), a master switch of allergic inflammation, plays an important role in the pathogenesis of allergic diseases. Although many compounds upregulate TSLP expression in vivo or in vitro, most of them are pollutants or toxicants. In the previous study, for the first time, we found that a steroid alkaloid derivative 02F04, which has a unique skeletal structure compared with other TSLP-inducing chemicals, significantly induced TSLP production in mouse keratinocytes. However, it is not investigated thoroughly that how 02F04 produces TSLP and why. In this study, we did a detailed investigation on the inducible effect and underlying molecular mechanism of 02F04 on TSLP production. We found that the peak time of TSLP mRNA level induced by 02F04 at 48 h led to a slow and continuous TSLP production in PAM212 cells. Besides, 02F04-induced TSLP production was significantly suppressed by inhibitors of Rho-associated protein kinase (ROCK), guanine nucleotide-binding protein subunit alpha q/11 (Gq/11) and extracellular signal-regulated kinase 1/2 (ERK1/2) at not only protein but also mRNA levels, and by siRNA-mediated knockdown of Gq or G11. This suggested that ROCK, Gq/11 and ERK1/2 signaling pathways were involved in 02F04-induced TSLP production. Increase in the level of p-ERK1/2 induced by 02F04 was suppressed by both inhibitors of ROCK and Gq/11, indicating that ROCK and Gq/11 molecules were located at the upstream of ERK1/2 to regulate 02F04-induced TSLP production. Gq/11 was located at the upstream of ROCK because the specific Gq/11 inhibitor of YM-254890 significantly reduced 02F04-induced actin stress fiber formation. Taken together, 02F04 upregulates a slow and continuous TSLP production through a novel Gq/11-ROCK-ERK1/2 signaling pathway. The thorough understanding the effect and mechanism of 02F04 on TSLP production is expected to supply it as a novel TSLP-regulating compound and a potential new tool for investigating the role of TSLP in allergic disorders.

Keywords: 02F04; Extracellular signal-regulated kinase 1/2; Gq/11; Rho-associated protein kinase; Thymic stromal lymphopoietin.