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SH-SY5Y_CB1R-antagonist_Proteomics

ABSTRACT: Cannabinoid receptor type 1 (CB1R) is one of the most abundant GPCRs in the brain, playing a critical role in neuronal viability. While CB1R activation is typically associated with neuroprotective effects, excessive or prolonged activation can be adverse. CB1R antagonists, therefore, have shown neuroprotective effects against neurotoxicity by CB1R overactivation. Unexpectedly, we found that CB1R antagonists rimonabant and AM251 induce cell death in human neuroblastoma SH-SY5Y cells under serum-free conditions, but not in serum-containing conditions. Phosphoproteomic analysis was performed to elucidate this molecular pathway. Phospho-EIF2AK3 (also known as PERK) was up-regulated in both rimonabant and AM251 treatments. PERK is a transducer of ER stress. Using western blotting it was also confirmed that the downstream effectors of PERK, eIF2α/ATF4/CHOP, were up-regulated, suggesting activation of the ER stress signal pathway PERK/eIF2α/ATF4/CHOP. In summary, cell death by CB1 antagonists under serum-free conditions results from ER stress response.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kosuke Kataoka

PROVIDER: PXD058518 | JPOST Repository | Tue Dec 03 00:00:00 GMT 2024

REPOSITORIES: jPOST

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Project description:Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-time PCR analyses confirmed that four of these factors, VEGF, FGF2, angiogenin and IL-8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGF regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGF promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGF expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGF gene, although its contribution to VEGF transcription appeared to be fairly modest. We also found that VEGF mRNA stability is increased in response to UPR activation, via activation of the AMP and p38MAP kinases, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGF protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGF expression at multiple levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer. We used microarrays to perform genome wide expression analysis in Daoy medulloblastoma cells to identify gene profiles that change in response to thapsigargin treatment which causes induction of the unfolded protein response.

Project description:Over the past several decades, corals worldwide have been affected by global warming, experiencing severe bleaching events that have often lead to coral death. The symbiotic Red Sea coral Stylophora pistillata is considered an opportunistic ‘r’ strategist, thriving in relatively unstable and unpredictable environments, and it is considered a stress-tolerant species. This study aimed to examine S. pistillata gene expression and to clarify the cellular pathways that are active during short-term heat stress caused by an increase from 24°C to 34°C over a 10-day period. Total RNA was extracted from heat-stressed coral fragments, labeled and hybridized against a designated S. pistillata custom microarray containing approximately 12,000 genes. Our results show that the heat stress reaction was sighted from 32°C and intensified significantly after 34°C treatment. Protein interaction networks of up- and down-regulated genes were constructed. The main clustering groups of up-regulated genes were ER stress and ER protein folding, cell cycle, ubiquitin-mediated proteolysis, cell death and cell death regulation and cellular stress response genes. These genes were enriched in cellular pathways related to the unfolded protein response (UPR) in the ER, ER-associated degradation (ERAD) and ubiquitin-mediated proteolysis. An analysis of the down-regulated genes yielded different clusters of genes related to extracellular matrix and actin organization, collagen, negative regulation of cell death and the Notch and Wnt signaling pathways. Genes encoding redox regulation proteins and molecular chaperones may be considered accurate “early warning genes”, while genes related to sensing and repairing DNA damage are severe heat-related genes. Here, we suggest that during short-term heat stress, S. pistillata might divert cellular energy into mechanisms such as UPR and ERAD at the expense of growth and biomineralization processes in an effort to recover from the stress.

			Action	DRS
	2-AM1h_750ng.raw	Raw
	2-D_750ng.raw	Raw
	2-R1h_750ng.raw	Raw
	240314.msf	Msf
	240314.pdResult	Other

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SH-SY5Y_CB1R-antagonist_Proteomics

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