Weibo Luo

Emerging roles of PKM2 in cell metabolism and cancer progression.

Increased conversion of glucose to lactate is a key feature of many cancer cells that promotes rapid growth. Pyruvate kinase M2 (PKM2) expression is increased and facilitates lactate production in cancer cells. Modulation of PKM2 catalytic activity also regulates the synthesis of DNA and lipids that are required for cell proliferation, and of NADPH that is required for redox homeostasis. In addition to its role as a pyruvate kinase, PKM2 also functions as a protein kinase and as a transcriptional coactivator. These biochemical activities are controlled by allosteric regulators and post-translational modifications of PKM2 that include acetylation, oxidation, phosphorylation, prolyl hydroxylation, and sumoylation. Given its pleiotropic effects on cancer biology, PKM2 represents an attractive target for cancer therapy.

Control of TH17/Treg Balance by Hypoxia-Inducible Factor 1

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

Pyruvate Kinase M2 Is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor 1

The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O(2) consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.

Hsp70 and CHIP Selectively Mediate Ubiquitination and Degradation of Hypoxia-inducible Factor (HIF)-1α but Not HIF-2α

Hypoxia-inducible factors (HIFs) are transcription factors that mediate adaptive responses to reduced oxygen availability. HIF-α subunits are stabilized under conditions of acute hypoxia. However, prolonged hypoxia leads to decay of HIF-1α but not HIF-2α protein levels by unknown mechanisms. Here, we identify Hsp70 and CHIP (carboxyl terminus of Hsc70-interacting protein) as HIF-1α-interacting proteins. Hsp70, through recruiting the ubiquitin ligase CHIP, promotes the ubiquitination and proteasomal degradation of HIF-1α but not HIF-2α, thereby inhibiting HIF-1-dependent gene expression. Disruption of Hsp70-CHIP interaction blocks HIF-1α degradation mediated by Hsp70 and CHIP. Inhibition of Hsp70 or CHIP synthesis by RNA interference increases protein levels of HIF-1α but not HIF-2α and attenuates the decay of HIF-1α levels during prolonged hypoxia. Thus, Hsp70- and CHIP-dependent ubiquitination represents a molecular mechanism by which prolonged hypoxia selectively reduces the levels of HIF-1α but not HIF-2α protein.

Hypoxia-inducible factor–dependent breast cancer–mesenchymal stem cell bidirectional signaling promotes metastasis

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) to promote metastasis. In a mouse orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to LNs and lungs in a HIF-dependent manner. Coculture of MSCs with BCCs augmented HIF activity in BCCs. Additionally, coculture induced expression of the chemokine CXCL10 in MSCs and the cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. CXCR3 expression was blocked in cocultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs cocultured with BCCs that did not express CXCR3 or HIFs. MSC coculture did not enhance the metastasis of HIF-deficient BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGFR1, respectively, in a HIF-dependent manner, and CXCL10 expression by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis.

Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis

Significance Cancer cells release from their cell surface membrane-lined microvesicles (MVs), which contain proteins, mRNAs, and microRNAs that can be taken up by other cells. We report that breast cancer cells exposed to decreased oxygen availability (hypoxia) increase their production of MVs, which stimulate invasion and metastasis by recipient breast cancer cells. Increased MV shedding by hypoxic cells requires expression of hypoxia-inducible factors (HIFs), which activate transcription of the RAB22A gene, and expression of the small GTPase RAB22A, which is a protein that localizes to budding MVs. Our results delineate a molecular mechanism by which hypoxia increases invasion and metastasis by stimulating MV shedding and provide further evidence that addition of HIF inhibitors to current treatment regimens may improve clinical outcome. Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.

Hypoxia-Inducible Factor 1 Mediates Increased Expression of NADPH Oxidase-2 in Response to Intermittent Hypoxia

Sleep‐disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro‐oxidant enzymes, especially NADPH oxidase‐2 (Nox2). Previous studies showed that (i) IH activates hypoxia‐inducible factor 1 (HIF‐1) in a ROS‐dependent manner and (ii) HIF‐1 is required for IH‐induced ROS generation, indicating the existence of a feed‐forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH‐induced expression of Nox2 is mediated by HIF‐1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild‐type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF‐1 activity through RNA interference or pharmacologic inhibition (digoxin or YC‐1) or by genetic knockout of HIF‐1α in MEFs. Increasing HIF‐1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF‐1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild‐type mice to IH (8 h/day for 10 days) up‐regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a+/− mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF‐1, ROS generation, and cardiovascular pathology in response to IH. J. Cell. Physiol. 226: 2925–2933, 2011.

Histone demethylase JMJD2C is a coactivator for hypoxia-inducible factor 1 that is required for breast cancer progression

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that play key roles in breast cancer biology. We hypothesized that interaction of HIF-1 with epigenetic regulators may increase HIF-1 transcriptional activity, and thereby promote breast cancer progression. We report that the histone demethylase jumonji domain containing protein 2C (JMJD2C) selectively interacts with HIF-1α, but not HIF-2α, and that HIF-1α mediates recruitment of JMJD2C to the hypoxia response elements of HIF-1 target genes. JMJD2C decreases trimethylation of histone H3 at lysine 9, and enhances HIF-1 binding to hypoxia response elements, thereby activating transcription of BNIP3, LDHA, PDK1, and SLC2A1, which encode proteins that are required for metabolic reprogramming, as well as LOXL2 and L1CAM, which encode proteins that are required for lung metastasis. JMJD2C expression is significantly associated with expression of GLUT1, LDHA, PDK1, LOX, LOXL2, and L1CAM mRNA in human breast cancer biopsies. JMJD2C knockdown inhibits breast tumor growth and spontaneous metastasis to the lungs of mice following mammary fat pad injection. Taken together, these findings establish an important epigenetic mechanism that stimulates HIF-1–mediated transactivation of genes encoding proteins involved in metabolic reprogramming and lung metastasis in breast cancer.

Trypsin and trypsin-like proteases in the brain: Proteolysis and cellular functions

Abstract.Several serine proteases including thrombin, tissue-type plasminogen activator and urokinase-type plasminogen activator have been well characterized in the brain. In this article, we review the brain-related trypsin and trypsin-like serine proteases. Accumulating evidence demonstrates that trypsin and trypsin-like serine proteases play very important roles in neural development, plasticity, neurodegeneration and neuroregeneration in the brain. Neuropsin is able to hydrolyze the extracellular matrix components by its active site serine, and regulates learning and memory in normal brain. The mutant neurotrypsin contributes to mental retardation in children. Neurosin seems to be involved in the pathogenesis of neurodegenerative disorders, like Alzheimer’s disease, Parkinson’s disease or multiple sclerosis. Although mesotrypsin/trypsin IV is also implicated in neurodegeneration, its functional significance still remains largely unknown. Particularly, mesotrypsin/trypsin IV, P22 and neurosin exert their physiological and pathological functions through activation of certain protease-activated receptors (PARs). In the brain, the presence of serpins controls the activity of serine proteases. Therefore, understanding the interaction among brain trypsin, serpins and PARs will provide invaluable tools for regulating normal brain functions and for the clinical treatment of neural disorders.

Hypoxia-inducible factor 1 is required for remote ischemic preconditioning of the heart

Significance Remote ischemic preconditioning (IPC) is a phenomenon in which brief cycles of limb ischemia and reperfusion, which can be induced in mice by clamping and unclamping the femoral artery and in human subjects by inflating and deflating a blood pressure cuff, result in protection of the heart against a prolonged episode of ischemia and reperfusion. The underlying mechanism may involve signals transmitted by the nervous system and by secreted factors, including interleukin-10. In this study, we demonstrate that the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is necessary and sufficient for remote IPC in mice and for induction of Il10 gene expression in cultured mouse myocytes. Strategies designed to induce HIF-1 activity may afford cardioprotection in patients undergoing cardiac bypass surgery. Both preclinical and clinical studies suggest that brief cycles of ischemia and reperfusion in the arm or leg may protect the heart against injury following prolonged coronary artery occlusion and reperfusion, a phenomenon known as remote ischemic preconditioning. Recent studies in mice indicate that increased plasma interleukin-10 (IL-10) levels play an important role in remote ischemic preconditioning induced by clamping the femoral artery for 5 min followed by 5 min of reperfusion for a total of three cycles. In this study, we demonstrate that remote ischemic preconditioning increases plasma IL-10 levels and decreases myocardial infarct size in wild-type mice but not in littermates that are heterozygous for a knockout allele at the locus encoding hypoxia-inducible factor (HIF) 1α. Injection of a recombinant adenovirus encoding a constitutively active form of HIF-1α into mouse hind limb muscle was sufficient to increase plasma IL-10 levels and decrease myocardial infarct size. Exposure of C2C12 mouse myocytes to cyclic hypoxia and reoxygenation rapidly increased levels of IL-10 mRNA, which was blocked by administration of the HIF-1 inhibitor acriflavine or by expression of short hairpin RNA targeting HIF-1α or HIF-1β. Chromatin immunoprecipitation assays demonstrated that binding of HIF-1 to the Il10 gene was induced when myocytes were subjected to cyclic hypoxia and reoxygenation. Taken together, these data indicate that HIF-1 activates Il10 gene transcription and is required for remote ischemic preconditioning.