Presented at the 2012 AAPM Annual Meeting « Back

Upregulation of Endogenous H2S Synthesis Enzyme Gene by Promoter Demethylation Contributes to Inflammatory Pain in Rats

Guang-Yin Xu, MD PhD, guangyin.xu@gmail.com, Soochow University, Suzhou, Jiangsu, PR China

Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator synthesized by cystathionine-beta-synthetase (CBS), is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms for role of CBS-H2S in peripheral nociceptive processing remain unknown. We showed here that peripheral inflammation significantly upregulated expression of H2S synthesizing enzyme CBS both at protein and mRNA levels in rat dorsal root ganglion (DRG). Using methylation specific PCR and bisulfate sequencing assays we provided evidence that CBS gene promoter region CpG island was remarkably demethylated in inflammatory rats when compared with control group. Furthermore, peripheral inflammation led to a significant upregulation of methyl-binding domain protein 4 (MBD4) and Gadd45a without marked alterations in expression of DNA methyltransferase 3a and 3b, and MBD2 and thymine DNA glycosylase. CBS inhibitors, hydroxylamine (HA) or AOAA, attenuated mechanical hyperalgesia and reversed hyperexcitability of DRG neurons in inflammatory rats. Intraplantar administration of NaHS or L-cysteine elicited mechanical hyperalgesia, in dose- and time-dependent fashions, in healthy rats. Application of NaHS in vitro enhanced excitability and led to a 35% increase in TTX-resistant (TTX-r) sodium current density of DRG neurons from healthy rats. H89, a protein kinase A inhibitor, blocked the potentiation of TTX-r current density. Our findings suggest that peripheral inflammation led to DNA demethylation in CBS gene promoter and upregulated CBS expression in DRGs. H2S appears to function as a novel nociceptive messenger through a PKA-mediated potentiation of TTX-r sodium channels, thus leading to inflammatory hyperalgesia. References: 1)Xu, G.Y., Winston, J.H., Shenoy, M., Zhou, S., Chen, J.D., and Pasricha, P.J. 2009. The endogenous hydrogen sulfide producing enzyme cystathionine-beta synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome. Mol Pain 2)Maeda, Y., Aoki, Y., Sekiguchi, F., Matsunami, M., Takahashi, T., Nishikawa, H., and Kawabata, A. 2009. Hyperalgesia induced by spinal and peripheral hydrogen sulfide: evidence for involvement of Cav3.2 T-type calcium channels. Pain 142:127-132. 3)Tang, G., Wu, L., and Wang, R. 2010. Interaction of Hydrogen Sulfide with Different Ion Channels. Clin Exp Pharmacol Physiol. 4)Rai, K., Huggins, I.J., James, S.R., Karpf, A.R., Jones, D.A., and Cairns, B.R. 2008. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell 135:1201-1212. 5)Ruddock-D'Cruz, N.T., Xue, J., Wilson, K.J., Heffernan, C., Prashadkumar, S., Cooney, M.A., Sanchez-Partida, L.G., French, A.J., and Holland, M.K. 2008. Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family dur 6)Xu, G.Y., and Huang, L.Y. 2002. Peripheral inflammation sensitizes P2X receptor-mediated responses in rat dorsal root ganglion neurons. J Neurosci 22:93-102.

Funding: This work was supported by grants from National Natural Science Foundation of China (81070884) and from Jiangsu Distinguished Professor Program (SR21500111).