Stephen C. Land

Hypoxia-inducible Factor 1α Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif

Tumors that form as a result of heightened mammalian target of rapamycin (mTOR) signaling are highly vascularized. This process of angiogenesis is regulated through hypoxia-inducible factor (HIF)-mediated transcription of angiogenic factors. It is recognized that inhibition of mTOR with rapamycin can diminish the process of angiogenesis. Our work shows that activation of mTOR by Ras homologue enriched in brain (Rheb) overexpression potently enhances the activity of HIF1α and vascular endothelial growth factor (VEGF)-A secretion during hypoxia, which is reversed with rapamycin. Mutants of Rheb, which do not bind guanine nucleotide (D60K, D60V, N119I, and D122N) and are unable to activate mTOR, inhibit the activity of HIF when overexpressed. We show that regulatory associated protein of mTOR (Raptor) interacts with HIF1α and requires an mTOR signaling (TOS) motif located in the N terminus of HIF1α. Furthermore, a mutant of HIF1α lacking this TOS motif dominantly impaired HIF activity during hypoxia and was unable to bind to the co-activator CBP/p300. Rapamycin treatments do not affect the stability of HIF1α and modulate HIF activity via a Von Hippel-Lindau (VHL)-independent mechanism. We demonstrate that the high levels of HIF activity in cells devoid of TSC2 can be reversed by treatments with rapamycin or the readdition of TSC2. Our work explains why human cancers with aberrant mTOR signaling are prone to angiogenesis and suggests that inhibition of mTOR with rapamycin might be a suitable therapeutic strategy.

A non-hypoxic, ROS-sensitive pathway mediates TNF-α-dependent regulation of HIF-1α

A non‐hypoxic, reactive oxygen species (ROS)‐sensitive pathway mediating tumor necrosis factor‐α (TNF‐α)‐dependent regulation of hypoxia‐inducible factor‐1α (HIF‐α) was investigated in vitro. TNF‐α mediated the translocation of HIF‐1α, associated with up‐regulating its activity under normoxia. Analysis of the mode of action of TNF‐α revealed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2 − ) and hydroxyl radical (OH). Antioxidants purported as prototypical scavengers of H2O2 and OH, attenuated TNF‐α‐induced HIF‐1α activation, and blockading NADPH‐oxidase by scavenging O2 − reduced the activity of HIF‐1α. Inhibition of the mitochondrion complex I abrogated TNF‐α‐dependent activation of HIF‐1α. Interrupting the respiratory chain reversed the excitatory effect of TNF‐α on HIF‐1α. These results indicate a non‐hypoxic pathway mediating cytokine‐dependent regulation of HIF‐1α in a ROS‐sensitive mechanism.

Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-α biosynthesis

Redox and ROS regulation of MAPK‐mediated TNF‐α biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS‐mediated TNF‐α secretion is redox‐dependent, NF‐κB‐sensitive and attenuated by N‐acetyl‐L‐cysteine (NAC) and other antioxidants. In alveolar epithelial cells, LPS induced a time‐ and dose‐dependent phosphorylation of MAPKp38. This was associated with the activation of MAPK‐activated protein kinase, which phosphorylated the small heat‐shock protein, Hsp27. MAPKp38 inhibition (SB‐203580) abrogated LPS‐induced TNF‐α production. MAPKERK blockade (PD‐98059) attenuated TNF‐α secretion, an effect synergistically amplified in the presence of SB‐203580. Regulation of NF‐κB by selective inhibitors revealed that this pathway is partially involved in regulating LPS‐mediated TNF‐α secretion. Whereas the proteasome inhibitor, MG‐132, had no effect on LPS‐mediated TNF‐α production, CAPE, sulfasalazine and SN‐50, a cell‐permeant NF‐κB inhibitor, attenuated but did not abrogate TNF‐α biosynthesis. LPS up‐regulated ROS, an effect abrogated by 4′‐hydroxy‐3′‐methoxy‐acetophenone and NAC, which reduced TNF‐α secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPKp38 pathway. ROS induced MAPKp38 phosphorylation and selective antioxidants, including the permeant GSH precursor, γ‐GCE, reduced ROS‐dependent MAPKp38 phosphorylation. These results indicate that the MAPK pathway and MAPK‐mediated regulation of TNF‐α production is redox‐dependent, GSH‐mediated and requires, at least in part, a NF‐κB/ROS‐sensitive mechanism.

Oxygen‐evoked Na+ transport in rat fetal distal lung epithelial cells

1 Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. 2 Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. 3 Reporter gene assays showed that no activation of the α‐ENaC (epithelial Na+ channel α‐subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. 4 Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6‐24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. 5 The increase in GNa thus coincides with activation of the α‐ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.

Alpha-melanocyte-related tripeptide, Lys-d-Pro-Val, ameliorates endotoxin-induced nuclear factor kappaB translocation and activation: evidence for involvement of an interleukin-1beta193-195 receptor antagonism in the alveolar epithelium.

The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB.

Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility

Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel–Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt–Hogg–Dubé syndrome. For this, we analysed a Birt–Hogg–Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt–Hogg–Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt–Hogg–Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the ‘Warburg effect’). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt–Hogg–Dubé-associated renal lesions.

Expression of Wild-Type CFTR Suppresses NF-κB-Driven Inflammatory Signalling

Background Mutation of the cystic fibrosis transmembrane-conductance regulator (CFTR) causes cystic fibrosis (CF) but not all CF aspects can easily be explained by deficient ion transport. CF-inflammation provides one example but its pathogenesis remains controversial. Here, we tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-κB activity. Methodology/Principal Findings In lung epithelial (H441) and engineered (H57) cell lines; we report that inflammatory markers are significantly suppressed by wild-type CFTR. Transient-transfection of wild-type CFTR into CFTR-naïve H441 cells, dose-dependently down-regulates both basal and Tumour Necrosis Factor-α evoked NF-κB activity when compared to transfection with empty vector alone (p<0.01, n>5). This effect was also observed in CFTR-naïve H57-HeLa cells which stably express a reporter of NF-κB activity, confirming that the CFTR-mediated repression of inflammation was not due to variable reporter gene transfection efficiency. In contrast, H57 cells transfected with a control cyano-fluorescent protein show a significantly elevated basal level of NF-κB activity above control. Initial cell seeding density may be a critical factor in mediating the suppressive effects of CFTR on inflammation as only at a certain density (1×105 cells/well) did we observe the reduction in NF-κB activity. CFTR channel activity may be necessary for this suppression because the CFTR specific inhibitor CFTRinh172 significantly stimulates NF-κB activity by ∼30% in CFTR expressing 16HBE14o− cells whereas pharmacological elevation of cyclic-AMP depresses activity by ∼25% below baseline. Conclusions/Significance These data indicate that CFTR has inherent anti-inflammatory properties. We propose that the hyper-inflammation found in CF may arise as a consequence of disrupted repression of NF-κB signalling which is normally mediated by CFTR. Our data therefore concur with in vivo and in vitro data from Vij and colleagues which highlights CFTR as a suppressor of basal inflammation acting through NF-κB, a central hub in inflammatory signalling.

Birt–Hogg–Dubé syndrome is a novel ciliopathy

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Redox signaling-mediated regulation of lipopolysaccharide-induced proinflammatory cytokine biosynthesis in alveolar epithelial cells.

The regulation of cytokine gene transcription and biosynthesis involves the reduction-oxidation (redox)-sensitive nuclear factor-kappaB (NF-kappaB), whose activation is mediated by an upstream kinase that regulates the phosphorylation of inhibitory-kappaB (IkappaB). It was hypothesized that lipopolysaccharide (LPS)-induced biosynthesis of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in vitro is regulated by redox equilibrium. In alveolar epithelial cells, we investigated the role of L-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inhibits glutathione oxidized disulfide reductase, pyrrolidine dithiocarbamate (PDTC), an antioxidant/prooxidant thiuram, and N-acetyl-L-cysteine (NAC), an antioxidant and GSH precursor, in regulating LPS-induced cytokine biosynthesis and IkappaB-alpha/NF-kappaB signaling. BSO blockaded the phosphorylation of IkappaB-alpha, reduced its degradation, and inhibited NF-kappaB activation, besides augmenting LPS-mediated biosynthesis of cytokines. BCNU up-regulated LPS-induced release of cytokines, an effect associated with partial phosphorylation/degradation of IkappaB-alpha and inhibition of the DNA binding activity. PDTC, which partially affected LPS-induced IkappaB-alpha phosphorylation/degradation, otherwise blockading NF-kappaB activation, reduced LPS-dependent up-regulation of cytokine release. Pretreatment with BSO did not abolish the NAC-dependent reduction of LPS-induced cytokine release, despite the fact that NAC marginally amplified IkappaB-alpha phosphorylation/degradation and suppressed NF-kappaB activation. These results indicate that cytokines are redox-sensitive mediators and that the IkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially implicated in mediating redox-dependent regulation of LPS-induced release of proinflammatory cytokines.

Oxygen-sensing pathways and the development of mammalian gas exchange

Abstract Oxygen-sensing pathways have been extensively explored in the context of homeostatic responses to hypoxic episodes; however, little is known of their involvement in the morphogenesis of respiratory structures (mitochondria, placenta, lung) during development in utero. This review identifies four essential loci where oxygen signalling pathways may cue the development of respiratory structures as: (i) mitochondrial biogenesis coupled with muted oxidative function dependent on the hypoxia-sustained production of NO; (ii) the generation of oxygen gradients which drive trophoblast differentiation and the formation of the chorionic gas exchange interface of the placenta; (iii) the proliferation and epithelial/endothelial differentiation of mesenchyme during the initiation of lung morphogenesis; and (iv) the regulation of epithelial fluid secretion/absorption in the lung. The identification of these oxygen-regulated developmental stages clarifies the close association between oxygen availability, reactive oxygen species and the morphogenesis of gas exchange structures and bears with it the implication that these pathways set the scope for aerobic metabolic performance throughout life.