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RTKs are recycled back to the plasma membrane via early endosomes and are delivered into either trans-Golgi networks or lysosomes for degradation via late endo-somes. How might hypoxia affect early endosome recycling and RTK trafficking from the late endosome into the trans-Golgi networks?

Finally, EGFR has recently been reported to stabilize a membrane-associated sodium-glucose transporter, SGLT1 (ref. 13), inde-pendently of its kinase function. Repression of early endosome fusion could increase cell surface EGFR expression. Hypoxia might also, conceivably, stabilize SGLT1 and empower cancer cell survival under hypoxia.

The newly discovered link between hypoxia and RTK signaling1 sets the stage for more exciting discoveries in the future.

1. Wang, Y. et al. Nat. Med. 15, 319–324 (2009).2. Harris, A.L. Nat. Rev. Cancer 2, 38–47 (2002).3. Gordan, J.D. & Simon, M.C. Curr. Opin. Genet. Dev. 17,

71–77 (2007).4. Denko, N.C. Nat. Rev. Cancer 8, 705–713 (2008).5. Semenza, G.L. Nat. Rev. Cancer 3, 721–732 (2003).6. Citri, A. & Yarden, Y. Nat. Rev. Mol. Cell Biol. 7, 505–

516 (2006).7. Mendelsohn, J. & Baselga, J. Oncogene 19, 6550–

6565 (2000).8. Cunningham, D. et al. N. Engl. J. Med. 351, 337–345

(2004).9. Ji, H. et al. Cancer Cell 9, 485–495 (2006).10. Franovic, A. et al. Proc. Natl. Acad. Sci. USA 104,

13092–13097 (2007).11. Horiuchi, H. et al. Cell 90, 1149–1159 (1997).12. Ohh, M. et al. Nat. Cell Biol. 2, 423–427 (2000).13. Weihua, Z. et al. Cancer Cell 13, 385–393 (2008).

degradation of RTKs. Hypoxia or loss of VHL can induce HIF activation, leading to enhanced RTK signaling by delaying early endosome fusion (Fig. 1).

It is plausible that this link between the oxygen-sensing pathway and delayed endo-cytosis may potentiate a broad spectrum of signaling events downstream of RTKs and other membrane receptors. It will be important to further determine the biologi-cal effect of downregulation of rabaptin-5 by HIF in vivo in animal models and to see whether HIF-1 behaves similarly to HIF-2, which was the focus of most of the experi-ments of Wang et al.1 Another open question is whether upregulation of HIF modulates other membrane proteins that undergo endocytosis-mediated turnover, especially those involved in oxygen consumption and cellular metabolism.

therefore decreases degradation of internal-ized EGFR in many types of cancer cells1. They further showed that HIF transcription-ally repressed rabaptin-5 expression, which delayed early endosome fusion and inhibited EGFR degradation1. Restored expression of rabaptin-5 reversed the effect.

The findings identify a general role of the oxygen-sensing pathway in endocytosis and

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Figure 1 Oxygen starvation feeds EGF. Wang et al. report that hypoxia-activated HIF inhibits rabaptin-5 expression, delays early endosome fusion and prolongs the activation of EGFR. EGF-stimulated EGFR is negatively regulated by the Rab5–rabaptin-5–mediated early endosome fusion, leading to lysosome-mediated EGFR degradation. Hypoxia-activated HIF binds to the hypoxia response element (HRE) of the RABEP1 promoter to inhibit transcription. Downregulation of rabaptin-5 delays Rab5–rabaptin-5–mediated early endosome fusion and therefore stabilizes the activation of EGFR.

Intolerant of glucose and gasping for oxygenFiona M Gribble

Findings in knockout mice indicate that hypoxia-sensitive pathways modulate the glucose-sensing machinery of pancreatic beta cells. Conditions that mimic hypoxia severely impair glucose-stimulated insulin release.

Fiona M. Gribble is at the Cambridge Institute for

Medical Research and Department of Clinical

Biochemistry, Addenbrooke’s Hospital, Hills Road,

Cambridge CB2 0XY, UK.

e-mail: fmg23@cam.ac.uk

Since the appearance of oxygen in the earth’s atmosphere, life has evolved to maximize energy yields from environmental nutrients, using this molecule as an electron acceptor. So great is our reliance on oxygen for energy production that cells have devised several layers of adaptive responses that switch on when oxygen levels fall and protect against the otherwise drastic effects of such hypoxia on cell function and survival.

Electrically active cells including neurons, muscle cells and endocrine cells, for exam-ple, express ATP-sensitive potassium (KATP) channels that open when ATP concentrations fall, thus dampening further electrical activ-ity and acutely reducing energy consump-tion. Longer-term reprogramming events in hypoxic tissues are triggered by the hypoxia inducible transcription factor (HIF) pathway, which not only increases a cell’s capacity to generate energy in the absence of oxygen but also stimulates local capillary growth to improve future oxygen delivery.

Three independent studies now link both of these adaptive systems1–3. The works examine the effect of HIF activation in insu-

lin-producing beta cells in the pancreas. Beta cells normally use KATP channels to monitor the rate of ATP production, which they use as a measure of the availability of glucose in the bloodstream. Activation of HIF in beta cells, however, results in diversion of the energy production pathways downstream of glucose—ultimately leading to impaired ATP production, reduced insulin secretion and whole-body glucose intolerance. The findings may explain why insulin release is impaired in inadequately oxygenated pancreatic islet transplants4. They also raise the possibility that HIF activation may contribute to the defective insulin secretion observed in people with type 2 diabetes.

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hydroxylated forms of HIF-α for proteasomal destruction (Fig. 1a)10. Work in a variety of tissues has shown that HIF-α hydroxylation is impaired during hypoxia, probably as a result of both reduced oxygen availability and raised levels of reactive oxygen species, and that the nonhydroxylated HIF-α is pro-tected against VHL-mediated degradation. Under hypoxia conditions, HIF-α therefore becomes available to dimerize with its part-ner, the aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF-1β), and to switch on genes that contain hypoxia responsive elements.

The inactivation of VHL in beta cells resulted in increased amounts of HIF-1α and increased expression of glycolytic enzymes and lactate dehydrogenase (Fig. 1b). Interestingly, although metabolic flux through glycolysis was enhanced in VHL-deficient beta cells, the diversion of metabolites away from the mito-chondria resulted in a phenotype of impaired glucose tolerance and drastically reduced glu-cose-stimulated insulin release1–3. To prove that the changes resulted from activation of HIF-1α, and not from effects on alternative targets of VHL, the researchers showed that inactivation of HIF-1α in beta cells, although not itself causing a noticeable metabolic phe-notype, prevented glucose intolerance in VHL-deficient mice1,3.

Because the activation of HIF-1α in beta cells apparently has such detrimental effects on insulin release, a leading question is why should beta cells express components of the HIF pathway at all? Perhaps beta cells, like other cells, simply need a mechanism to pro-tect against hypoxia, and, as beta cells have a very poor regenerative capacity, it may be important to preserve their longer-term viability even if the temporary consequence is a collapse of whole-body glucose homeo-stasis.

However, work on other tissue systems would suggest that HIF-1α activity in beta cells might be influenced by glucose-depen-dent fluctuations in the metabolic rate even during normoxia, for example via altered mitochondrial oxygen consumption, hyper-glycemia-associated generation of reactive oxygen species11 or metabolic intermediates such as succinate12. One potential advantage of the consequent diversion of metabolites away from mitochondria may act in con-cert with uncoupling protein-2 to protect beta cells against excessive free radical gen-eration13. It is also conceivable that HIF-1α activity may have a physiological role in determining the working glucose set point of a beta cell. The analysis of beta cell–targeted HIF-1α–knockout mice under conditions of

chondrial respiration, generating elevated amounts of ATP, NADPH and citric acid cycle intermediates (Fig. 1a). The increased ratio of ATP to ADP, arising largely from mitochon-drial oxidation, initiates the first phase of insulin release by closing plasma membrane KATP channels, thereby enabling beta cells to depolarize and fire action potentials7. The prolonged second phase of insulin release is achieved by additional—but incompletely characterized—signaling pathways involv-ing ATP, NADPH and other mitochon-drial metabolites, which further enhance the release of insulin-containing secretory granules8.

In light of the tight link between islet metabolism and insulin secretion, it is per-haps surprising that the new studies show not only that components of the HIF pathway are expressed in pancreatic beta cells9 but also that activation of HIF-1α (the most widely expressed form of HIF-1α) switches beta cell metabolism toward glycolysis, thereby damp-ening insulin secretion1–3.

To experimentally increase HIF-α activity in beta cells, each of the three research teams used transgenic mouse technology to inacti-vate the von Hippel Lindau protein (VHL) specifically in pancreatic beta cells. VHL is a ubiquitin ligase that normally keeps cel-lular amounts of HIF-α low by targeting

HIF-α was first recognized for its capacity to turn on erythropoietin production during hypoxia5, providing a molecular explanation for the observation that people living at high altitudes have higher than average counts of red blood cells. It is now recognized that HIF-α has a more generalized role in hypoxia sensing and that hypoxia responsive elements coordinate transcription of a range of genes that contribute to the longer-term adaptation to hypoxia6. The metabolic consequence is an enhanced rate of glycolysis, resulting from increased synthesis of glycolytic enzymes and lactate dehydrogenase. This enzyme maintains a high flux through glycolysis in the absence of oxygen by preventing NADH accumulation and shunting glycolytic end products away from the mitochondria and out of the cell in the form of lactate.

Although the majority of cell types do their utmost to defend their ATP stores in the face of insults such as hypoxia and hypoglyce-mia—which would otherwise put a dent in ATP production—insulin-releasing beta cells in the pancreatic islets actually harness the fluctuating metabolic rate to couple the availability of glucose to their rate of insulin secretion.

Beta cell metabolism increases several fold at higher circulating glucose concentrations as a result of increased glycolytic and mito-

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Figure 1 Glucose-stimulated insulin secretion from pancreatic beta cells is triggered by glycolytic and mitochondrial metabolism, resulting in the generation of ATP, KATP channel closure and Ca2+ entry. Secretion is further amplified by products of mitochondrial respiration. (a) VHL normally targets HIF-1α for proteasomal degradation, but in its absence (b) HIF-1α levels rise. Three new studies1–3 show that the absence of HIF-1α triggers the expression of a range of enzymes that enhance glycolysis at the expense of mitochondrial metabolism. The diversion of nutrients away from mitochondria interferes with glucose-triggered insulin release, resulting in a whole-body phenotype of glucose intolerance.

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3. Zehetner, J. et al. Genes Dev. 22, 3135–3146 (2008).

4. Moritz, W. et al. FASEB J. 16, 745–747 (2002).5. Semenza, G.L. & Wang, G.L. Mol. Cell. Biol. 12, 5447–

5454 (1992).6. Semenza, G.L. Nat. Rev. Cancer 3, 721–732 (2003).7. Ashcroft, F.M. J. Clin. Invest. 115, 2047–2058

(2005).8. Wiederkehr, A. & Wollheim, C.B. Endocrinology 147,

2643–2649 (2006).9. Gunton, J.E. et al. Cell 122, 337–349 (2005).10. Maxwell, P.H. et al. Nature 399, 271–275 (1999).11. Taylor, C.T. Biochem. J. 409, 19–26 (2008).12. Selak, M.A. et al. Cancer Cell 7, 77–85 (2005).13. Affourtit, C. & Brand, M.D. Biochim. Biophys. Acta

1777, 973–979 (2008).

That glucose intolerance can result from both over- and underactivity of the HIF path-way in beta cells probably reflects the precise interplay between beta cell metabolism and insulin secretion. Such apparently conflicting findings highlight the potential pitfalls of try-ing to target the HIF pathway therapeutically in beta cells without a greater understanding of its role and regulation.

1. Cantley, J. et al. J. Clin. Invest. 119, 125–135 (2009).

2. Puri, S. et al. Diabetes 58, 433–441 (2009).

metabolic stress may help to answer some of these questions.

These three studies raise the question of whether HIF-1α activation contributes to beta cell dysfunction in diabetes. In con-trast to the new findings1–3, a previous study found that expression of ARNT is actually reduced in islets from humans with type 2 diabetes9, and that ARNT inactivation in beta cells impairs insulin secretion, probably as a result of an interplay with other transcription factors regulating beta cell function.

When integrins fail to integrateAndrés Hidalgo & Paul S Frenette

Three studies implicate Kindlin-3, a molecule that mediates signaling through integrins, in a rare disorder characterized by spontaneous bleeding and susceptibility to infection (pages 300–305, 306–312 and 313–318).

Andrés Hidalgo and Paul S. Frenette are at Mount

Sinai School of Medicine, Departments of Medicine

and Gene and Cell Medicine, Black Family Stem

Cell Institute, Immunology Institute, 1 Gustave Levy

Place, Box 1079, New York, New York 10029, USA. e-mail: paul.frenette@mssm.edu

Integrins, by switching from a resting low- affinity state to an active high-affinity confor-mation, can rapidly make blood cells sticky. Tight regulation of this major class of cell adhesion molecules ensures normal blood flow and leukocyte trafficking and mediates rapid adhesion during events such as the for-mation of a platelet plug after vessel injury.

Several naturally occurring mutations can inactivate the platelet β3 integrin, leading to Glanzmann’s thrombasthenia, characterized by spontaneous bleeding due to defective platelet aggregation, whereas individuals with homozygous mutations in the gene encoding β2 integrin (CD18) have recurrent infectious complications due to leukocyte adhesion defi-ciency type 1 (LAD-I)1. More recently, indi-viduals showing both Glanzmann’s-like and LAD-like symptoms have been described in which the expression of integrins is normal but integrin activity on leukocytes and platelets is defective2. This rare but devastating syndrome is termed LAD-I variant or LAD-III.

This syndrome was thought to result from altered signal transduction downstream from G protein–coupled receptors (GPCRs), as their chemokine ligands, major integrin activators, are unable to activate integrins on leukocytes and platelets isolated from people

affected with this condition. One potential culprit has been the diacylglycerol-regu-lated guaninenucleotide exchange factor I (CALDAG-GEF1, official symbol RASGRP2); mice lacking this protein show an LAD-III–like phenotype3, and a homozygous splice junction mutation in the Caldaggef1 gene has been observed in people with the disease4.

In this issue of Nature Medicine, three different groups of researchers point to a more likely culprit5–7. They provide strong evidence that a deficiency in Kindlin-3, a molecule downstream of GPCRs that helps coax integrins into an active conformation, underlies LAD-III5,6,7.

The Kindlin family of proteins, comprised of three members, is named after Theresa Kindler, who first described a congenital skin blistering condition that combined clinical features of hereditary epidermoly-sis bullosa and poikiloderma congenitale8. The Kindler syndrome is caused by loss-of-function mutations in Kindlin-1 (official symbol FERMT1), which participates in the integrin-dependent anchorage of the actin cytoskeleton within focal adhesions. In mice, deficiency in Kindlin-2 (Kindlin2−/−) leads to a failure of the early embryo to implant in the uterus resulting from impaired integrin activation in embryonic stem cells9.

Kindlin-3 is expressed in blood cells, and mice lacking the molecule (Kindlin3−/−) have defects in platelet aggregation and resistance to arterial thrombosis7. These observations led to the question of whether Kindlin-3 is involved in the LAD-III syndrome.

The report by Svensson et al.5 indeed describes KINDLIN3 mutations in three subjects diagnosed with LAD-III resulting in undetectable expression of the protein. Whereas two of these subjects also presented a mutation in CALDAGGEF1, no such muta-tion was found in the third subject.

In the study by Malinin et al.6, two sib-lings showing classical LAD-III symptoms were found to harbor a distinct mutation in the KINDLIN3 gene and had no Kindlin-3 protein expression, whereas expression and function of CALDAG-GEF1 was normal. Importantly, complementation studies in both reports revealed that the enforced expression of Kindlin-3, but not CALDAG-GEF1, restored the adhesive deficiencies in cells lines derived from the subjects.

Interestingly, the two subjects studied by Malinin et al.6 had osteopetrosis (a condi-tion producing abnormal thickening of the bones) that was suggested to have originated from an enhanced capacity of mesenchy-mal stem cells to generate bone. However, because Kindlin-3 expression is restricted to the hematopoietic lineage10, the cause of the osteopetrosis might lie with osteoclasts, hematopoietic cells mediating αvβ3 integrin–dependent bone resorption11.

Experiments in mice by Moser et al.7 delved into the mechanism, providing insight into how Kindlin-3 deficiency might lead to disease. As with the LAD-III human cells, leukocytes from mice deficient in Kindlin-3 showed impaired adhesion to and spreading on ligands for β2 integrins. This translated