Date of Award
Doctor of Philosophy
Regulators of G-protein signaling (RGS) are a multifunctional and highly diverse group of proteins that negatively regulate G-protein coupled receptor (GPCR) signaling pathways. The common mechanism of RGS is to act as GTPase accelerating proteins (GAP) to accelerate the hydrolysis of active GTP bound G proteins and terminate the actions of the associated GPCR. In addition to the traditional function of inhibiting G-protein signaling, recent studies have highlighted the role of RGS proteins in modulating GPCRs in GAP-independent way. There are more than 30 RGS proteins, and depending upon cell/tissue type, they interact and associate with different G proteins and GPCRs to modulate various physiological functions. RGS17, a member of the RGS-RZ subfamily, commonly targets GTP bound Gαz, Gαi, and Gαo for hydrolysis and signal termination. RGS17 is abundantly expressed in the central nervous system and is highly associated with opioid, dopamine and cannabinoid receptors in the brain. RGS17 is also upregulated in many malignant tumors such as lung, prostate and breast cancers. Analysis of whole cochlea transcriptome data from our lab revealed higher levels of RGS17 in the cochlea after cisplatin treatment. This highlights a possible role of RGS17 (and probably other RGS proteins) in cisplatin ototoxicity. Activation of endogenous, otoprotective GPCRs such as adenosine (A1AR) and cannabinoid (CB2) receptor is beneficial for promoting protection against cisplatin-induced hearing loss. Taking all this together, the underlying hypothesis for this study is that cisplatin could possibly mediate ototoxicity by increasing the expression of RGS17, which reduces the otoprotective effect of endogenous receptors such as cannabinoid receptor 2. The main objective of the study is to examine the expression and function of RGS17 in the cochlea and determine if inhibition of RGS17 could protect against cisplatin ototoxicity.The expression of RGS17 was observed in the both in vitro and in vivo models of cochlear cell types. Immunofluorescence study, western blot analysis, and RT-qPCR results showed the presence of RGS17 in UB\OC-1, as well as Wistar rat cochlea; expression levels increased after cisplatin treatment. To determine the role of RGS17 in hearing, first, it was overexpressed in the cochlea using adenoviral vector that was found to significantly increase ABR threshold shifts and decrease ABR Wave I amplitude. Conversely, knockdown of RGS17 (by siRGS17) decreased cisplatin-induced elevations in ABR thresholds along with increased wave I amplitude and latency. Furthermore, siRGS17 pretreatment prevented cisplatin-mediated synapse loss at inner hair cells. This indicates inhibition of RGS17 can preserve the functional and physiological integrity of the cochlea, which is essential for hearing. Cochleae that were treated with siRGS17, followed by cisplatin, showed fewer TUNEL-positive cells and reduced loss of Outer hair cells (OHC) as compared to cisplatin-treated rats. Moreover, overexpression of RGS17 increased the ratio of the transcription factors, pSTAT1/pSTAT3, which may indicate initiation of the apoptotic pathway. Moreover, UB\OC-1 cells treated with Celastrol, a RGS17 inhibitor, showed an increase in cell viability against cisplatin toxicity. In addition to apoptosis, overexpression of RGS17 also elevated ROS production and oxidative stress. But, the inhibition of RGS17 attenuated the cisplatin-induced increase in transcripts for oxidative and inflammatory stress markers, such as NOX3, iNOS, KIM1, TNF-α, and COX2, whereas the mRNA level of antioxidant genes such as Nrf2 and SOD2 were increased. Activation of CB2 via JWH-015 (a CB2 agonist) prior to cisplatin administration significantly reduced the cisplatin-induced elevated levels of RGS17, while knockdown of CB2 increased RGS17 expression in the cochlea. siRGS17 treatment boosted endogenous CB2R-Gα expression. Additionally, cisplatin decreased the expression of Gαi/o and Gαz in vitro, but the activation of CB2 increased the expression of these G proteins. Furthermore, JWH-015 treatment alleviated RGS17-dependent cell death. This study suggests that RGS17 could serve as a mediator of cisplatin ototoxicity by reducing the duration of active CB2R-G protein signaling, which normally suppresses cochlear oxidative stress, inflammation and hair cell apoptosis, and thereby preserves normal hearing. These data also indicate the existence of tonic reciprocal inhibition between RGS17 and CB2 mediated via the G proteins. Thus, we propose that RGS17 inhibitors could serve as an effective treatment against cisplatin ototoxicity when used alone and can potentiate the actions of CB2 agonists when used in combination therapy against cisplatin-induced hearing loss.
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