Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression

We have previously demonstrated the roles of RhoA, Rac1, and Cdc42 in hypoxia-driven angiogenesis. However, the role of oncogenes in hypoxia signaling is poorly understood. Given the importance of Rho proteins in the hypoxic response, we hypothesized that Rho family members could act as mediators of hypoxic signal transduction. We investigated the cross-talk between hypoxia and oncogene-driven signal transduction pathways and explored the role of Rac1 on hypoxia-induced hypoxia-inducible factor (HIF)-1α and VEGF expression. Since the phosphatidylinositol 3'-kinase (PI3K) pathway is involved in signal transduction of many oncogenes, we explored the role of PI3K on Rac1-mediated expression of HIF-1α and VEGF in hypoxia. We showed that LY-294002, a PI3K inhibitor, suppressed HIF-1α and VEGF induction under hypoxic conditions by up to 50%. Activation of Rac1 resulted in an upregulation of hypoxia-induced HIF-1α expression, which was blocked by LY-294002. These data suggested that Rac1 is an intermediate in the PI3K-mediated induction of HIF-1α. Interestingly, there was a significant downregulation of the tumor suppressor genes p53 and von Hippel-Lindau tumor suppressor (VHL) in cells expressing a constitutively active form of Rac1. Rac1-mediated inhibition of p53 and VHL could therefore be implicated in the upregulation of HIF-1α expression.     

Podocytes protect glomerular endothelial cells from hypoxic injury via deSUMOylation of HIF-1α signaling

Hypoxia can cause severe tubulointerstitial injury and peritubular capillary loss. However, hypoxia-induced injury in glomerular capillaries is far milder than tubulointerstitium, but the reason for this difference is unclear. We hypothesized that the phenomenon is due to the protective crosstalk among intrinsic glomerular cells. To mimic the microenvironment and investigate the crosstalk process temporally, we established co-culture models of glomerular endothelial cells (GEnCs) with podocytes or with mesangial cells. We found that podocytes rather than mesangial cells prevented GEnCs from injury and hypoxia-induced apoptosis and promoted migration and angiogenesis of GEnCs under hypoxic conditions. We then identified that increased activation of the hypoxia inducible factor 1α (HIF-1α) pathway as the major mechanism enabling podocytes to protect GEnCs against hypoxia. HIF-1α stabilization during hypoxia is known to be dependent on SUMO-specific protease 1 (SENP1)-mediated deSUMOylate modifications. Therefore, we further targeted deSUMOylation, regulated by SENP1, by short hairpin RNA (shRNA) knockdown of SENP1 mRNA in vitro and measured expression of HIF-1α and its downstream gene VEGF in hypoxic podocytes. Our results showed that SENP1 was essential for HIF-1α deSUMOylation in podocytes. The blockade of deSUMOylation by SENP1 shRNA successfully abolished the activation of HIF-1α signaling and consequently suppressed the protective effects of podocytes on GEnCs. In conclusion, we demonstrate for the first time that hypoxia may promote HIF-1α stabilization and activation by increasing SENP1 expression in podocytes, which induce GEnCs survival and angiogenesis to resist hypoxia. Thus, deSUMOylation of HIF-1α signaling is a potentially novel therapeutic target for treating hypoxic renal disorders.     

Hypoxia-inducible factor-1α mediates oral squamous cell carcinoma invasion via upregulation of α5 integrin and fibronectin

With progressive and rapid growth of malignant tumors, cancer cells in an ischemic condition are expected to develop an increased potential for local invasive growth. To address this hypothesis, we first examined the effect of hypoxia on the invasiveness of oral squamous cell carcinoma (OSCC) cells using the Matrigel invasion assay. We then investigated the effect of hypoxia on the protein and mRNA expression of α5 integrin and fibronectin, which are major factors involved in tumor cell invasion. We showed that (i) hypoxia increased the invasiveness of OSCC cells, (ii) α5 integrin and fibronectin protein and mRNA expression levels were increased in OSCC cells under hypoxic conditions, (iii) hypoxia stimulated autocrine secretion of fibronectin in OSCC cells, (iv) administration of siRNA_HIF-1α caused a significant decrease in α5 integrin and fibronectin protein, confirming that HIF-1α plays a role in their induction, and (v) siRNA_HIF-1α abrogated hypoxia-induced cell invasion. Collectively, these data suggest that hypoxia promotes OSCC cell invasion that is elicited by HIF-1α-dependent α5 integrin and fibronectin induction.     

Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.     

Hypoxia inducing factor-1α regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia

Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1α is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1α (HIF-1α) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1α protein. Hypoxia- and DEF-induced HIF-1α activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1α inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1α using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1α inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1α inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells.     

Aldose reductase inhibition prevents hypoxia-induced increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) by regulating 26 S proteasome-mediated protein degradation in human colon cancer cells

The development of intratumoral hypoxia, a hallmark of rapidly progressing solid tumors, renders tumor cells resistant to chemotherapy and radiation therapy. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes the reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mouse xenografts by inhibiting the NF-κB-dependent activation of oxidative stress-mediated inflammatory and carcinogenic markers. However, the role of AR in mediating hypoxic stress signals is not known. We therefore investigated the molecular mechanisms by which AR inhibition prevents the hypoxia-induced human colon cancer cells growth and invasion. Our results indicate that AR inhibition by the pharmacological inhibitor fidarestat or ablation by AR-specific siRNA prevents hypoxia-induced proliferation of HT29, SW480, and Caco-2 colon cancer cells. Furthermore, hypoxia-induced increase in the level of HIF-1α in colon cancer cells was significantly decreased by AR inhibition. During hypoxic conditions, treatment of HT29 cells with the AR inhibitor fidarestat significantly decreased the expression of vascular endothelial growth factor, a down target of HIF-1α, at both mRNA and protein levels and also prevented the activation of PI3K/AKT, GSK3β, Snail, and lysyl oxidase. Furthermore, inhibition of hypoxia-induced HIF-1α protein accumulation by AR inhibition was abolished in the presence of MG132, a potent inhibitor of the 26 S proteasome. In addition, AR inhibition also prevented the hypoxia-induced inflammatory molecules such as Cox-2 and PGE2 and expression of extracellular matrix proteins such as MMP2, vimentin, uPAR, and lysyl oxidase 2. In conclusion, our results indicate that AR mediates hypoxic signals, leading to tumor progression and invasion.     

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.     

Blocking OLFM4/HIF-1α axis alleviates hypoxia-induced invasion,epithelial–mesenchymal transition,and chemotherapy resistance in non-small-cell lung cancer

Hypoxia is a common biological hallmark of solid cancers, which has been proposed to be associated with oncogenesis and chemotherapy resistance. The purpose of the present study was to investigate the role and underlying mechanisms of olfactomedin 4 (OLFM4) in the hypoxia-induced invasion, epithelial–mesenchymal transition (EMT), and chemotherapy resistance of non-small-cell lung cancer (NSCLC). We observed dramatically upregulated expression of OLFM4 in several NSCLC cell lines, and this effect was more pronounced in A549 and H1299 cells. In addition, our data revealed that OLFM4 expression was remarkably increased in both A549 and H1299 cells under hypoxic microenvironment, accompanied by enhanced levels of hypoxia-inducible factor (HIF)-1α protein. The HIF-1α level was elevated in response to hypoxia, resulting in the regulation of OLFM4. Interestingly, OLFM4 was a positive regulator of hypoxia-driven HIF-1α production. Moreover, depletion of OLFM4 modulated multiple EMT-associated proteins, as evidenced by the enhanced E-cadherin levels along with the diminished expression of N-cadherin and vimentin in response to hypoxia, and thus blocked invasion ability of A549 and H1299 cells following exposure to hypoxia. Furthermore, ablation of OLFM4 accelerated the sensitivity of A549 cells to cisplatin under hypoxic conditions, implying that OLFM4 serves as a key regulator in chemotherapeutic resistance under hypoxia. In conclusion, OLFM4/HIF-1α axis might be a potential therapeutic strategy for NSCLC.     

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.     

p53 Gene deficiency promotes hypoxia-induced pulmonary hypertension and vascular remodeling in mice

Chronic hypoxia induces pulmonary arterial remodeling, resulting in pulmonary hypertension and right ventricular hypertrophy. Hypoxia has been implicated as a physiological stimulus for p53 induction and hypoxia-inducible factor-1α (HIF-1α). However, the subcellular interactions between hypoxic exposure and expression of p53 and HIF-1α remain unclear. To examine the role of p53 and HIF-1α expression on hypoxia-induced pulmonary arterial remodeling, wild-type (WT) and p53 knockout (p53KO) mice were exposed to either normoxia or hypoxia for 8 wk. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as measured by the ratio of the right ventricle to the left ventricle plus septum weights, and vascular remodeling. However, the right ventricular systolic pressures, the ratio of the right ventricle to the left ventricle plus septum weights, and the medial wall thickness of small vessels were significantly greater in the p53KO mice than in the WT mice. The p53KO mice had lower levels of p21 and miR34a expression, and higher levels of HIF-1α, VEGF, and PDGF expression than WT mice following chronic hypoxic exposure. This was associated with a higher proliferating cell nuclear antigen expression of pulmonary artery in p53KO mice. We conclude that p53 plays a critical role in the mitigation of hypoxia-induced small pulmonary arterial remodeling. By interacting with p21 and HIF-1α, p53 may suppress hypoxic pulmonary arterial remodeling and pulmonary arterial smooth muscle cell proliferation under hypoxia.     

A novel regulation of VEGF expression by HIF-1α and STAT3 in HDM2 transfected prostate cancer cells

On the basis of increasing roles for HDM2 oncoprotein in cancer growth and progression, we speculated that HDM2 might play a major role in hypoxia-induced metastatic process. For verification of this hypothesis, wild-type LNCaP prostate cancer cells and HDM2 transfected LNCaP-MST (HDM2 stably transfected) cells were studied. The data obtained from our experiments revealed that the HDM2 transfected LNCaP-MST cells possessed an ability to multiply rapidly and show distinct morphological features compared to non-transfected LNCaP cells. During exposures to hypoxia HDM2 expression in the LNCaP and LNCaP-MST cells was significantly higher compared to the normoxic levels. The LNCaP-MST cells also expressed higher levels of HIF-1α (hypoxia-inducible factor-1α) and p-STAT3 even under the normoxic conditions compared to the non-transfected cells. The HIF-1α and p-STAT3 expressions were increased several fold when the cells were subjected to hypoxic conditions. The HIF-1α and p-STAT3 protein expressions observed in HDM2 transfected LNCaP-MST cells were 20 and 15 folds higher, respectively, compared to the non-transfected wild-type LNCaP cells. These results demonstrate that HDM2 may have an important regulatory role in mediating the HIF-1α and p-STAT3 protein expression during both normoxic and hypoxic conditions. Furthermore, the vascular endothelial growth factor (VEGF) expression that is typically regulated by HIF-1α and p-STAT3 was also increased significantly by 136% (P < 0.01) after HDM2 transfection. The overall results point towards a novel ability of HDM2 in regulating HIF-1α and p-STAT3 levels even in normoxic conditions that eventually lead to an up-regulation of VEGF expression.     

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5 μmol/L) and CAY10585 (10 μmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30 μmol/L) and LY294002 (30 μmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.