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.     

A Pharmacological Activator of AMP-Activated Protein Kinase Protects Hypoxic Neurons in a Concentration-Dependent Manner

5′adenosine monophosphate-dependent protein kinase (AMPK) is a member of metabolite-sensing kinase family which plays an important role in intracellular energy metabolism, particularly in the hypoxic neurons process. However, the effect of AMPK activation on hypoxic neurons remains controversial. In the present study, we report that the effect of AMPK activation induced by pretreatment with 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR) in neurons using the hypoxic model in vitro. The level of AMPK activation, the neuronal viability, and the levels of two important cytoskeleton proteins were analyzed during the oxygen deprivation. The AMPK activation was increased with the elevation of the AICAR concentration in hypoxic neurons. Moreover, the AMPK activation induced by AICAR protected neurons against death from hypoxic deprivation and that strongly depended on the extent of the AMPK activation. The AMPK activation at specific range protects hypoxic neurons, but the protective effect of AMPK activation disappeared when the AMPK was over-activated by AICAR. The result from an AMPK inhibitor, Compound C, in hypoxic neurons further proves the neuroprotective effect of AICAR.     

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.     

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.     

MafG controls the hypoxic response of cells by accumulating HIF-1alpha in the nuclei

We identified MafG as a protein that interacts with HIF-1alpha, a key factor in hypoxic response, using the yeast two-hybrid system. Interaction between MafG and HIF-1alpha was confirmed by surface plasmon resonance and by translocation to the nucleolus with the NoLS signal. A knockdown of MafG reduced erythropoietin (EPO) mRNA levels as well as luciferase reporter activity with the hypoxia response element. The knockdown of MafG did not change total HIF-1alpha protein, but reduced the accumulation of HIF-1alpha in the nuclei. These results suggest that MafG regulates the hypoxic response of cells by detaining HIF-1alpha in the nuclei.     

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 signaling pathway

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)–GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 pathway.     

HIF-2alpha expression in human fetal paraganglia and neuroblastoma: relation to sympathetic differentiation, glucose deficiency, and hypoxia

Solid tumors are frequently necrotic and hypoxic due to poor vascularization. Tumor cells adapt to hypoxia by modulating their phenotype. Key players in this process are the hypoxia-inducible factors (HIF-1alpha to 3alpha). HIFs are also expressed during normal development; for example, HIF-2alpha is specifically expressed and appears to be involved in the development of the murine sympathetic nervous system (SNS). Here, we demonstrate that HIF-2alpha protein is selectively present in human fetal week 8.5 SNS paraganglia. Neuroblastoma is derived from SNS precursors. In a subset of neuroblastomas, a spontaneous neuronal to neuroendocrine differentiation occurs in areas adjacent to necrotic zones. As HIF-2alpha activity has been associated not only with hypoxic but also with hypoglycemic conditions, we have investigated putative effects of hypoxia, glucose depletion, and HIF-2alpha on the neuroblastoma phenotype. HIF-2alpha was detected in hypoxic and in well-oxygenized neuroblastoma cells and tissue, presumably reflecting their embryonic features. With regard to differentiation, hypoxic cells lost their neuronal/neuroendocrine features and gained marker gene expression associated with an immature, neural crest-like phenotype. Low glucose potentiated the effect of hypoxia. These findings suggest that poorly vascularized neuroblastomas become immature and maintain a more aggressive phenotype, which possibly could involve a sustained stabilization and activation of HIF-2alpha.     

AMPK-mediated autophagy is a survival mechanism in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia

The present study was designed to investigate (i) the role of AMPK activation in inducing autophagy in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia, and (ii) whether autophagy offers a survival advantage under these harsh conditions. Low androgen and low oxygen are two co-existing conditions frequently found in prostate cancer tissue following surgical or medical castration. In LNCaP cells, androgen deprivation and hypoxia together boosted AMPK activation to a higher level than that seen with either condition alone. The augmented AMPK response was associated with improved viability and the induction of autophagy. These observations suggest that a threshold of AMPK activity has to be attained in order to trigger autophagy, since neither androgen deprivation nor hypoxia by itself was capable of pushing AMPK activity past that threshold. Beclin-1 was identified as a potential downstream target of AMPK in turning on the autophagic cascade. If autophagy was blocked by chemical inhibition or RNA interference of key regulators, e.g., AMPK or beclin-1, more cells would die by apoptosis. The occurrence of autophagy is thus a survival mechanism for androgen-dependent prostate cancer cells to escape from an androgen-deprived and hypoxic subsistence.     

Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9 degrees C). In contrast, anoxia exposure (0.01 mg O2/l at 8 degrees C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is alpha2, beta1B, gamma2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory gamma2-subunit increased in the heart during anoxia. In the brain, expression of the alpha1-, alpha2-, and gamma1-subunits decreased with anoxia exposure, but expression of the gamma2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.