Aggregated ursolic acid, a natural triterpenoid, binds to CD36 for inducing interleukin-1β release from murine peritoneal macrophages

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Triterpenoids are ubiquitously distributed throughout the plant kingdom, with some being used as anti-tumor and anti-inflammatory agents in many Asian countries. It has been reported that ursolic acid (UA; 3β-hydroxy-12-urs-12-en-28-oic acid), a pentacyclic triterption carcinogenesis in rodents. Further, UA attenuated the expression of iNOS and COX-2 via NF-κB repression in stimulated RAW264.7 mouse macrophages (1). In contrast, You et al. reported that UA induced NO and TNF-αproduction via NF-κB activation when that cell line was not stimulated (2). Along a similar line, we recently reported that UA promoted the release of MIF via ERK2 activation (3). In the present study, we examined the effects of UA on the production of IL-1β, IL-6, MIF, and TNF-α in resident peritoneal macrophages (pMϕ) from female ICR mice. UA increased the protein release of IL-1β, IL-6, and MIF, but not TNF-α, into culture media in dose- and time-dependent manners. In addition, it strikingly induced activation of p38 MAPK and ERK1/2, but not JNK1/2, together with that of upstream kinases (MKK3/6 and MEK1/2). IL-1β release induced by UA was significantly inhibited by the inhibitors of p38 MAPK, MEK1/2, ABC transporter, and caspase-1, however, not by the inhibitor of JNK1/2 or the MAPK negative inhibitor. UA also induced intracellular reactive oxygen species (ROS) generation for IL-1β production, which was suppressed by an antioxidant. Pretreatment with an anti-CD36 antibody, but not antibodies for SR-A, CXCL16, and CD68, significantly suppressed IL-1β release, while surface plasmon resonance assay results showed that UA was bound to CD36 on the cell surface of macrophages. We also noted that the level of IL-1β released from UA-treated pMϕ from CD36-deficient mice was markedly lower than that from those of wild-type mice. Interestingly, UA was found to aggregate in culture media and the aggregates were suggested to be responsible for IL-1β production. Notably, the ability for inducing IL-1β was observed only with UA and not 6 other UA-related triterpenes, including oleanolic acid and glycyrrhetinic acid. Moreover, IL-1β protein was spontaneously released from the pMϕ of ICR mice administrated with UA. Together, our results indicate that aggregated UA is recognized, at least in part, by CD36 on macrophages for generating ROS, thereby activating p38 MAPK, ERK1/2, and caspase-1, as well as releasing IL-1β protein via the ABC transporter. In conclusion, treatment of non-stimulated, normal tissues with UA may trigger pro-inflammatory and tumor promoting effects. References: 1) Suh, N., et al. Cancer Res. 58:723, 1998; 2) You, H. J., et al. FEBS Lett. 509: 156, 2001; 3) Ikeda, Y., et al., Biochem. Pharmacol. 70: 1497-1505, 2005.