Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

The Early Activation of PI3K Strongly Enhances the Resistance of Cortical Neurons to Hypoxic Injury via the Activation of Downstream Targets of the PI3K Pathway and the Normalization of the Levels of PARP Activity, ATP, and NAD+

Phosphatidylinositol 3-kinase (PI3K) plays several important roles in neuronal survival. Activation of the pathway is essential for the neuroprotective mechanisms of materials that shield neuronal cells from many stressful conditions. However, there have been no reports to date about the effect of the direct activation of the pathway in hypoxic injury of neuronal cells. We investigated whether the direct activation of the PI3K pathway inhibits neuronal cell death induced by hypoxia. Primary cultured cortical neurons (PCCNs) were exposed to hypoxic conditions (less than 1 mol% O₂) and/or treated with PI3K activator. Hypoxia reduced the viability of PCCNs in a time-dependent manner, but treatment with PI3K significantly restored viability in a concentration-dependent manner. Among the signaling proteins involved in the PI3K pathway, those associated with survival, including Akt and glycogen synthase kinase-3β, were decreased shortly after exposure to hypoxia and those associated with cell death, including BAX, apoptosis-induced factor, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were increased. However, treatment with PI3K activator normalized the expression levels of those signaling proteins. PARP activity and levels of ATP and NAD⁺ altered by hypoxia were also normalized with direct PI3K activation. All these findings suggest that direct and early activation is important for protecting neuronal cells from hypoxic injury.     

低氧诱导因子-1的转录活性调控及其信号传导

低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)是氧平衡调控相关的转录因子.依赖HIF-1的基因表达调控系统广泛影响葡萄糖代谢、细胞增殖、凋亡和血管发生,与机体低氧适应、胚胎发育、各种缺血性疾病及肿瘤相关.HIF-1自身活性调节是低氧应答基因表达调控的中心环节.调控主要发生在源于Ras的两条信号途径：Ras/Raf/MEK介导的HIF-1反式激活功能调控,PI(3)K/Akt依赖的HIF-1alpha蛋白稳定性调控.这两个信号传导途径分别独立又协调地调控着HIF-1的转录活性.     

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial–mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.     

Role of hypoxia‐induced fibronectin‐integrin β1 expression in embryonic stem cell proliferation and migration: Involvement of PI3K/Akt and FAK

Cell migration is largely dependent on integrin (IN) binding to the extracellular matrix, and several signaling pathways involved in these processes have been shown to be modified by hypoxia. Therefore, the aim of this study was to determine the influence of hypoxia on fibronectin (FN) and IN β1 expression in mouse embryonic stem cells (mESCs) and their signaling pathways to modulate proliferation. FN and IN β1 expression were significantly increased in hypoxic mESCs by 24 h. Hypoxia also increased cell attachment, which was accompanied by concomitant increases in the binding level of FN and IN β1. Hypoxia‐induced FN expression was mediated by increased phosphatidylinositol 3 kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) phosphorylation, and hypoxia‐inducible factor‐1α (HIF‐1α) expression. Moreover, under hypoxic conditions, focal adhesion kinase (FAK) and Src phosphorylation were increased in a time‐dependent fashion; these increases were blocked by IN β1 antibody. In addition, the hypoxia induced increase of F‐actin distribution and cell migration (activation of matrix metalloproteinase‐2 and ‐9) was inhibited by IN β1 antibody. Indeed, hypoxia increased the level of cell‐cycle regulatory protein and DNA synthesis. In conclusion, hypoxia increases the proliferation and migration of mESCs via FN‐IN β1 production through the PI3K/Akt, mTOR, and HIF‐1α pathways, followed by FAK activation. J. Cell. Physiol. 226: 484–493, 2011. © 2010 Wiley‐Liss, Inc.