Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

Cold-inducible protein RBM3 mediates hypothermic neuroprotection against neurotoxin rotenone via inhibition on MAPK signalling.

Mild hypothermia and its key product, cold-inducible protein RBM3, possess robust neuroprotective effects against various neurotoxins. However, we previously showed that mild hypothermia fails to attenuate the neurotoxicity from MPP+ , one of typical neurotoxins related to the increasing risk of Parkinson disease (PD). To better understand the role of mild hypothermia and RBM3 in PD progression, another known PD-related neurotoxin, rotenone (ROT) was utilized in this study. Using immunoblotting, cell viability assays and TUNEL staining, we revealed that mild hypothermia (32°C) significantly reduced the apoptosis induced by ROT in human neuroblastoma SH-SY5Y cells, when compared to normothermia (37°C). Meanwhile, the overexpression of RBM3 in SH-SY5Y cells mimicked the neuroprotective effects of mild hypothermia on ROT-induced cytotoxicity. Upon ROT stimulation, MAPK signalling like p38, JNK and ERK, and AMPK and GSK-3ß signalling were activated. When RBM3 was overexpressed, only the activation of p38, JNK and ERK signalling was inhibited, leaving AMPK and GSK-3ß signalling unaffected. Similarly, mild hypothermia also inhibited the activation of MAPKs induced by ROT. Lastly, it was demonstrated that the MAPK (especially p38 and ERK) inhibition by their individual inhibitors significantly decreased the neurotoxicity of ROT in SH-SY5Y cells. In conclusion, these data demonstrate that RBM3 mediates mild hypothermia-related neuroprotection against ROT by inhibiting the MAPK signalling of p38, JNK and ERK.

Identification and characterization of the druggable kinase targets of olmesartan and its analogues from a systematic kinase-chemical interaction profile in atherosclerosis.

Olmesartan (OL) is the pharmacologically active metabolite of Olmesartan medoxomil (OM), an FDA-approved angiotensin II receptor antagonist for administrating cardiovascular diseases. The drug has been found to have potential effects on diverse protein kinase signaling involved in the pathogenesis of atherosclerosis, either by directly inhibiting the hub kinases or by indirectly modulating marginal members in the signaling pathways. In the present study, we computationally model the kinase-chemical Interaction Profile between six OL-related chemicals (i.e. OL, OM, Valsartan [VL], Losartan [LS], Candesartan [CD] and Telmisartan [TL]) and 23 human protein kinases in atherosclerosis. The profile is analyzed systematically at molecular level to identify unexpected kinase targets for OL. There is a good consistence between co-citation frequency and affinity scoring for the chemical association with kinase candidates; the OL and its analogs VL and LS exhibit a similar binding profile to the atherosclerosis kinase spectrum. It is suggested that the Ser/Thr-specific kinases PI3Kα and ROCK1 are potential druggable targets of OL for atherosclerosis therapy. As a paradigm, kinase assays reveal that the inhibitory potency of OL and Y-27632 (positive control) on ROCK1 is determined at micromolar level, while the OM (negative control) possesses no detectable activity for the kinase.

Cold-Inducible Protein RBM3 Protects UV Irradiation-Induced Apoptosis in Neuroblastoma Cells by Affecting p38 and JNK Pathways and Bcl2 Family Proteins.

Induced by hypothermia, cold-inducible protein RBM3 (RNA-binding protein motif 3), has been implicated in neuroprotection against various toxic insults such as hypoxia and ischemia. However, whether mild hypothermia and RBM3 prevent neural cells from UV irradiation-elicited apoptosis is unclear. In the present study, human neuroblastoma cell line SH-SY5Y was used as a cell model for neural cell death, and it was demonstrated that mild hypothermia protects SH-SY5Y cells from UV irradiation-induced apoptosis. However, the protective effect of mild hypothermia was abrogated when RBM3 was silenced. Conversely, the overexpression of RBM3 rescued SH-SY5Y cells from UV-induced apoptosis, as indicated by the decreased levels of cleaved caspase-3 and PARP, and increased cell survival. The analysis on the mechanism underlying RBM3-mediated neuroprotection against UV insult showed that RBM3 could substantially block the activation of p38 and JNK signaling pathways. In addition, the overexpression of RBM3 reduced the expression of pro-apoptotic proteins Bax and Bad, leaving the pro-survival protein Bcl-2 unaffected. In conclusion, RBM3 is the key mediator of mild hypothermia-related protection against UV in neuroblastoma cells, and the neuroprotective effect might be exerted through interfering with pro-apoptotic signaling pathways p38 and JNK and regulating pro-apoptotic proteins Bax and Bad.

RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143.

Nitric oxide (NO)-induced apoptosis in neurons is an important cause of neurodegenerative disease in humans. The cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults. However, whether RBM3 protects neural cells from NO-induced apoptosis is unclear. This study aimed to investigate the neuroprotective effect of RBM3 on NO-induced apoptosis in human SH-SY5Y neuroblastoma cells. Firstly, we demonstrated that mild hypothermia (32 °C) induces RBM3 expression and confers a potent neuroprotective effect on NO-induced apoptosis, which was substantially diminished when RBM3 was silenced by siRNA. Moreover, overexpression of RBM3 exhibited a strong protective effect against NO-induced apoptosis. Signaling pathway screening demonstrated that only p38 inhibition by RBM3 provided neuroprotective effect, although RBM3 overexpression could affect the activation of p38, JNK, ERK, and AKT signaling in response to NO stimuli. Notably, RBM3 overexpression also blocked the activation of p38 signaling induced by transforming growth factor-ß1. Furthermore, both RBM3 overexpression and mild hypothermia abolished the induction of miR-143 by NO, which was shown to mediate the cytotoxicity of NO in a p38-dependent way. These findings suggest that RBM3 protects neuroblastoma cells from NO-induced apoptosis by suppressing p38 signaling, which mediates apoptosis through miR-143 induction.