Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, USA.
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA,
Cell Physiol Biochem. 2022 Aug 19;56(4):367-381. doi: 10.33594/000000557.
BACKGROUND/AIMS: We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions.
INS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry.
HG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions.
Activation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.
背景/目的：我们最近报道称，在高糖（HG）条件下，胰腺β（INS-1 832/13）细胞中 NFκB（Rel A）p65 亚基的磷酸化（在 S536 处）增加。我们还表明，HG 诱导的 p65 的 S536 磷酸化是 CARD9 的调节作用的下游，因为 CARD9 表达的缺失显著减弱了β细胞中 HG 诱导的 p65 的 S536 磷酸化。当前研究的总体目标是确定胰岛β细胞在暴露于 HG 条件下，HG 诱导的 p65 磷酸化的潜在机制。
将 INS-1 832/13 细胞在低葡萄糖（LG；2.5 mM）或高葡萄糖（HG；20 mM）中孵育 24 小时，同时存在或不存在 G 蛋白异戊二烯化和激活的小分子抑制剂。使用市售（NE-PER）试剂盒从 INS-1 832/13 细胞中分离非核和核级分。通过蛋白质印迹和密度测定法定量 p65 亚基的 S536 磷酸化程度。
HG 诱导的 p65 磷酸化明显受到蛋白质异戊二烯化抑制剂（例如辛伐他汀和 L-788,123）的抑制。Tiam1-Rac1（例如 NSC23766）和 Vav2-Rac1（例如 Ehop-016）信号通路的药理学抑制对 HG 诱导的 p65 磷酸化几乎没有影响。然而，小分子化合物 EHT-1864 结合 Rac1 从而阻止 GDP/GTP 交换，显著抑制 HG 诱导的 p65 磷酸化，表明 Rac1 激活是 HG 介导的 p65 磷酸化所必需的。最后，EHT-1864 显著抑制了 INS-1 832/13 细胞在 HG 条件下暴露时 STAT3 的核结合，但不抑制总 p65。
Rac1 的激活，是 HG 诱导的 CARD9 激活的下游步骤，可能代表导致胰腺β细胞中 HG 诱导的 p65 的 S536 磷酸化和 STAT3 的核结合的级联反应中的必需信号步骤。这些研究的数据进一步证实了 Rac1 作为代谢应激诱导的胰岛β细胞功能障碍的介质的作用。
