RESUMO
Hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. We have previously reported that phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes HIF-1α nuclear accumulation and stimulates HIF-1 activity while lack of this modification triggers HIF-1α nuclear export and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ impairs HIF-1 activity by obstructing the formation of a HIF-1α/ARNT heterodimer. Investigation of these two antagonistic events by expressing double phospho-site mutants in HIF1A-/- cells under hypoxia revealed independent and additive phosphorylation effects that can create a gradient of HIF-1α subcellular localization and transcriptional activity. Furthermore, modification by CK1δ caused mitochondrial release of the non-nuclear HIF-1α form and binding to microtubules via its N-terminal domain. In agreement, endogenous HIF-1α could be shown to co-localize with mitotic spindle microtubules and interact with tubulin, both of which were inhibited by CK1δ silencing or inhibition. Moreover, CK1δ expression was necessary for equal partitioning of mother cell-produced HIF-1α to the daughter cell nuclei at the end of mitosis. Overall, our results suggest that phosphorylation by CK1δ stimulates the association of non-nuclear HIF-1α with microtubules, which may serve as a means to establish a symmetric distribution of HIF-1α during cell division under low oxygen conditions.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Quinases , Humanos , Mitose , Microtúbulos , Hipóxia , OxigênioRESUMO
Hypoxia is frequently encountered in the microenvironment of solid tumors. Hypoxia-inducible factors (HIFs), the main effectors of cell response to hypoxia, promote cancer cell survival and progression. HIF-1α, the oxygen-regulated subunit of HIF-1, is often correlated with oncogenesis and represents an attractive therapeutic target. We have previously reported that activation HIF-1α by ERK involves modification of two serine residues and masking of a nuclear export signal (NES), all inside a 43-amino acid domain termed ERK Targeted Domain (ETD). Overexpression of ETD variants including wild-type, phospho-mimetic (SE) or NES-less (IA) mutant forms caused HIF-1 inactivation in two hepatocarcinoma cell lines, while a phospho-deficient (SA) form was ineffective and acted as a sequence-specific negative control. To deliver these ETD forms directly into cancer cells, they were fused to the HIV TAT-sequence and produced as cell-permeable peptides. When the TAT-ETD peptides were added to the culture medium of Huh7 cells, they entered the cells and, with the exception of ETD-SA, accumulated inside the nucleus, caused mislocalization of endogenous HIF-1α to the cytoplasm, significant reduction of HIF-1 activity and inhibition of expression of specific HIF-1, but not HIF-2, gene targets under hypoxia. More importantly, transduced nuclear TAT-ETD peptides restricted migration, impaired colony formation and triggered apoptotic cell death of cancer cells grown under hypoxia, while they produced no effects in normoxic cells. These data demonstrate the importance of ERK-mediated activation of HIF-1 for low oxygen adaptation and the applicability of ETD peptide derivatives as sequence-specific HIF-1 and cancer cell growth inhibitors under hypoxia.
Assuntos
Apoptose/fisiologia , Peptídeos Penetradores de Células/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Hipóxia Celular , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Sinais de Exportação Nuclear/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Homologia de Sequência de Aminoácidos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Hypoxia inducible factor-1 (HIF-1) is the main transcriptional activator of the cellular response to hypoxia and an important target of anticancer therapy. Phosphorylation by ERK1 and/or ERK2 (MAPK3 and MAPK1, respectively; hereafter ERK) stimulates the transcriptional activity of HIF-1α by inhibiting its CRM1 (XPO1)-dependent nuclear export. Here, we demonstrate that phosphorylation by ERK also regulates the association of HIF-1α with a so-far-unknown interaction partner identified as mortalin (also known as GRP75 and HSPA9), which mediates non-genomic involvement of HIF-1α in apoptosis. Mortalin binds specifically to HIF-1α that lacks modification by ERK, and the HIF-1α-mortalin complex is localized outside the nucleus. Under hypoxia, mortalin mediates targeting of unmodified HIF-1α to the outer mitochondrial membrane, as well as association with VDAC1 and hexokinase II, which promotes production of a C-terminally truncated active form of VDAC1, denoted VDAC1-ΔC, and protection from apoptosis when ERK is inactivated. Under normoxia, transcriptionally inactive forms of unmodified HIF-1α or its C-terminal domain alone are also targeted to mitochondria, stimulate production of VDAC1-ΔC and increase resistance to etoposide- or doxorubicin-induced apoptosis. These findings reveal an ERK-controlled, unconventional and anti-apoptotic function of HIF-1α that might serve as an early protective mechanism upon oxygen limitation and promote cancer cell resistance to chemotherapy.
Assuntos
Apoptose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/metabolismo , Hipóxia Celular , Ativação Enzimática , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hexoquinase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Transporte Proteico , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Hypoxia-inducible factors (HIF) are master regulators of the response to hypoxia. Although several kinases are known to modify their oxygen sensitive HIF-α subunits or affect indirectly their function, little is known about the role of phosphatases in HIF control. To address this issue, a library containing siRNAs for the 25 known catalytic subunits of human phosphatases was used to screen for their effect on HIF transcriptional activity in HeLa cells. Serine-threonine phosphatase PPP3CA (calcineurin A, isoform a) was identified as the strongest candidate for a negative regulator of HIF activity. Indeed, independent silencing of PPP3CA expression stimulated HIF transcriptional activity under hypoxia, without increasing the protein levels of HIF-1α or HIF-2α. Overexpression of a constitutively active PPP3CA form, but not its catalytically inactive counterpart, inhibited HIF activity and expression of HIF target genes but did not affect HIF-1α or HIF-2α expression. These results were phenocopied by treatment with the ionophore ionomycin, that activates endogenous PPP3CA. The effect of ionomycin was mediated by PPP3CA as it was largely abolished by PPP3CA silencing. Furthermore, ionomycin enhanced the down-regulation of HIF activity by wild-type PPP3CA overexpression. Overall, our results suggest the involvement of PPP3CA in fine-tuning the HIF-dependent transcriptional response to hypoxia.
Assuntos
Calcineurina/metabolismo , Regulação para Baixo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transcrição Gênica , Calcineurina/genética , Ativação Enzimática , Inativação Gênica , Células HeLa , Humanos , Ionomicina/farmacologiaRESUMO
Hypoxia inducible factor 2 (HIF-2) is a transcriptional activator implicated in the cellular response to hypoxia. Regulation of its inducible subunit, HIF-2α (also known as EPAS1), involves post-translational modifications. Here, we demonstrate that casein kinase 1δ (CK1δ; also known as CSNK1D) phosphorylates HIF-2α at Ser383 and Thr528 in vitro We found that disruption of these phosphorylation sites, and silencing or chemical inhibition of CK1δ, reduced the expression of HIF-2 target genes and the secretion of erythropoietin (EPO) in two hepatic cancer cell lines, Huh7 and HepG2, without affecting the levels of HIF-2α protein expression. Furthermore, when CK1δ-dependent phosphorylation of HIF-2α was inhibited, we observed substantial cytoplasmic mislocalization of HIF-2α, which was reversed upon the addition of the nuclear protein export inhibitor leptomycin B. Taken together, these data suggest that CK1δ enhances EPO secretion from liver cancer cells under hypoxia by modifying HIF-2α and promoting its nuclear accumulation. This modification represents a new mechanism of HIF-2 regulation that might allow HIF isoforms to undertake differing functions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caseína Quinase Idelta/metabolismo , Eritropoetina/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Núcleo Celular/metabolismo , Inativação Gênica , Células HeLa , Células Hep G2 , Humanos , Carioferinas/metabolismo , Neoplasias Hepáticas/genética , Mutação/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Fatores Estimuladores Upstream/metabolismo , Proteína Exportina 1RESUMO
Hypoxia inducible factor-1 (HIF-1) supports survival of normal cells under low oxygen concentration and cancer cells in the hypoxic tumor microenvironment. This involves metabolic reprogramming via upregulation of glycolysis, downregulation of oxidative phosphorylation and, less well documented, effects on lipid metabolism. To investigate the latter, we examined expression of relevant enzymes in cancer cells grown under hypoxia. We show that expression of acylglycerol-3-phosphate acyltransferase 2 (AGPAT2), also known as lysophosphatidic acid acyltransferase ß (LPAATß), was upregulated under hypoxia and this was impaired by siRNA-mediated knockdown of HIF-1α. Moreover, a sequence of the AGPAT2 gene promoter region, containing 6 putative Hypoxia Response Elements (HREs), activated transcription of a reporter gene under hypoxic conditions or in normoxic cells over-expressing HIF-1α. Chromatin immunoprecipitation experiments confirmed binding of HIF-1α to one of these HREs, mutation of which abolished hypoxic activation of the AGPAT2 promoter. Knockdown of AGPAT2 by siRNA reduced lipid droplet accumulation and cell viability under hypoxia and increased cancer cell sensitivity to the chemotherapeutic etoposide. In conclusion, our findings demonstrate that AGPAT2, which is mutated in patients with congenital generalized lipodystrophy and over-expressed in different types of cancer, is a direct transcriptional target of HIF-1, suggesting that upregulation of lipid storage by HIF-1 plays an important role in adaptation and survival of cancer cells under low oxygen conditions.
Assuntos
Aciltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metabolismo dos Lipídeos/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Etoposídeo/farmacologia , Glicerofosfolipídeos/biossíntese , Células HEK293 , Células HeLa , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Elementos de Resposta , Transdução de Sinais , Ativação Transcricional , Triglicerídeos/biossínteseRESUMO
Hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor of the bHLH-PAS family, is comprised of HIF-1α, which is inducible by hypoxia and ARNT or HIF-1ß, which is constitutively expressed. HIF-1 is involved in cellular homeostasis under hypoxia, in development and in several diseases affected by oxygen availability, particularly cancer. Since its expression is positively correlated with poor outcome prognosis for cancer patients, HIF-1 is a target for pharmaceutical therapy. We have previously shown that male germ cell Rac GTPase activating protein (MgcRacGAP), a regulator of Rho proteins which are principally involved in cytoskeletal organization, binds to HIF-1α and inhibits its transcriptional activity. In this work, we have explored the mechanism of the MgcRacGAP-mediated HIF-1 inactivation. We show that the Myo domain of MgcRacGAP, which is both necessary and sufficient for HIF-1 repression, binds to the PAS-B domain of HIF-1α. Furthermore MgcRacGAP competes with ARNT for binding to the HIF-1α PAS-B domain, as shown by in vitro binding pull down assays. In mammalian cells, ARNT overexpression can overcome the MgcRacGAP-mediated inhibition and MgcRacGAP binding to HIF-1α in vivo inhibits its dimerization with ARNT. We additionally present results indicating that MgcRacGAP binding to HIF-1α is specific, since it does not affect the transcriptional activity of HIF-2, a close evolutionary relative of HIF-1 also involved in hypoxia regulation and cancer. Our results reveal a new mechanism for HIF-1 transcriptional activity regulation, suggest a novel hypoxia-cytoskeleton link and provide new tools for selective HIF-1 inhibition.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia , Transcrição Gênica , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Western Blotting , Células Cultivadas , Citoesqueleto , Proteínas Ativadoras de GTPase/genética , Humanos , Imunoprecipitação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Ativação TranscricionalRESUMO
Adaptation to hypoxia involves hypoxia-inducible transcription factors (HIFs) and requires reprogramming of cellular metabolism that is essential during both physiological and pathological processes. In contrast to the established role of HIF-1 in glucose metabolism, the involvement of HIFs and the molecular mechanisms concerning the effects of hypoxia on lipid metabolism are poorly characterized. Here, we report that exposure of human cells to hypoxia causes accumulation of triglycerides and lipid droplets. This is accompanied by induction of lipin 1, a phosphatidate phosphatase isoform that catalyzes the penultimate step in triglyceride biosynthesis, whereas lipin 2 remains unaffected. Hypoxic upregulation of lipin 1 expression involves predominantly HIF-1, which binds to a single distal hypoxia-responsive element in the lipin 1 gene promoter and causes its activation under low oxygen conditions. Accumulation of hypoxic triglycerides or lipid droplets can be blocked by siRNA-mediated silencing of lipin 1 expression or kaempferol-mediated inhibition of HIF-1. We conclude that direct control of lipin 1 transcription by HIF-1 is an important regulatory feature of lipid metabolism and its adaptation to hypoxia.
Assuntos
Hipóxia Celular/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidato Fosfatase/biossíntese , Triglicerídeos/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Fator 1 Induzível por Hipóxia/genética , Fosfatidato Fosfatase/genética , Regiões Promotoras Genéticas , Triglicerídeos/biossíntese , Triglicerídeos/genética , Regulação para CimaRESUMO
BACKGROUND: Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). METHODS: ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle α-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-3H]scopolamine binding studies were performed in order to assess M3 muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[3H]thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. RESULTS: Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of α-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. CONCLUSIONS: Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways.
Assuntos
Acetilcolina/administração & dosagem , Carbacol/administração & dosagem , Proteínas Contráteis/biossíntese , Proteínas Contráteis/efeitos dos fármacos , Agonistas Muscarínicos/administração & dosagem , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Traqueia/citologia , Acetilcolina/farmacologia , Animais , Carbacol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Agonistas Muscarínicos/farmacologia , Coelhos , Fatores de TempoRESUMO
Airway smooth muscle cells (ASMCs) participate in tissue remodeling characteristic of airway inflammatory diseases like asthma. Inflammation and hypoxia pathways are often interconnected and the regulatory subunit of the hypoxia inducible factor, HIF-1α, has been recently shown to be induced by cytokines. Here we investigate the effect of individual or combined treatment of ASMCs with the inflammatory mediator TNFα and/or hypoxia on the expression of HIF-1α, HIF-1 targets and inflammation markers. TNFα enhances HIF-1α protein and mRNA levels, under both normoxia and hypoxia. TNFα-mediated induction of HIF-1α gene transcription is repressed by inhibition of the NF-κB pathway. Despite the up-regulation of HIF-1α protein, the transcription of HIF-1 target genes remains low in the presence of TNFα at normoxia and is even reduced at hypoxia. We show that the reduction in HIF-1 transcriptional activity by TNFα is due to inhibition of the interaction of HIF-1α with ARNT and subsequent blocking of its binding to HREs. Comparison between hypoxia and TNFα for their effects on the expression of inflammatory markers shows significant differences: hypoxia up-regulates the expression of IL-6, but not RANTES or ICAM, and reduces the induction of VCAM by TNFα. Finally, ex vivo treatment of rabbit trachea strips with TNFα increases HIF-1α protein levels, but reduces the expression of HIF-1 targets under hypoxia. Overall, TNFα induces HIF-1α mRNA synthesis via an NF-κB dependent pathway but inhibits binding of HIF-1α to ARNT and DNA, while hypoxia and TNFα have distinct effects on ASMC inflammatory gene expression.
Assuntos
Brônquios/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Traqueia/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima , Animais , Brônquios/citologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Células Cultivadas , Marcação de Genes , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Coelhos , Traqueia/citologia , Regulação para Cima/genéticaRESUMO
Hypoxia-inducible factor-1, a heterodimer of alpha (HIF-1α) and beta (HIF-1ß or ARNT) subunits, is a major regulator of the transcriptional response to hypoxia. However, HIF-1α, the oxygen-regulated subunit, also exerts nontranscriptional functions through interaction with proteins other than ARNT. We have previously shown that the subcellular localization and protein interactions of HIF-1α are controlled by ERK-mediated phosphorylation at Ser641/643. When HIF-1α is modified at these sites, it is nuclear, binds to ARNT, interacts with nucleophosmin 1 (NPM1) and activates transcription of hypoxia-target genes. On the contrary, unmodified HIF-1α is bound by chromosomal region maintenance 1 (CRM1), exits the nucleus and, via its association with mortalin, is targeted to the mitochondria to form an antiapoptotic complex. To further characterize the latter function, recombinant fragments of HIF-1α and mortalin were used for in vitro binding assays and immunoprecipitation experiments to map the respective binding sites and show that their interaction is direct and functional. We could also show that embelin, a natural product and known inhibitor of the mortalin-p53 interaction, also disrupts the mortalin-HIF-1α association and, furthermore, removes unmodified HIF-1α from mitochondria. Mitochondrial dissociation of HIF-1α, either by embelin or overexpression of a HIF-1α peptide harbouring the mortalin binding site, under stress conditions leads to mitochondrial localization of the pro-apoptotic protein B-cell lymphoma 2-associated X protein (Bax) and induction of apoptosis. We suggest that when ERK activity is low under hypoxia, binding of HIF-1α to mortalin inhibits mitochondrial recruitment of Bax and protects cells from apoptotic cell death.
Assuntos
Hipóxia , Mitocôndrias , Humanos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Hipóxia Celular/fisiologia , Mitocôndrias/metabolismo , Hipóxia/metabolismo , Apoptose , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
The Hypoxia Inducible Factor 1 (HIF-1) plays a major role in the cellular response to hypoxia by regulating the expression of many genes involved in adaptive processes that allow cell survival under low oxygen conditions. Adaptation to the hypoxic tumor micro-environment is also critical for cancer cell proliferation and therefore HIF-1 is also considered a valid therapeutical target. Despite the huge progress in understanding regulation of HIF-1 expression and activity by oxygen levels or oncogenic pathways, the way HIF-1 interacts with chromatin and the transcriptional machinery in order to activate its target genes is still a matter of intense investigation. Recent studies have identified several different HIF-1- and chromatin-associated co-regulators that play important roles in the general transcriptional activity of HIF-1, independent of its expression levels, as well as in the selection of binding sites, promoters and target genes, which, however, often depends on cellular context. We review here these co-regulators and examine their effect on the expression of a compilation of well-characterized HIF-1 direct target genes in order to assess the range of their involvement in the transcriptional response to hypoxia. Delineating the mode and the significance of the interaction between HIF-1 and its associated co-regulators may offer new attractive and specific targets for anticancer therapy.
Assuntos
Fator 1 Induzível por Hipóxia , Neoplasias , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/genética , Regiões Promotoras Genéticas , Neoplasias/genética , Cromatina , Oxigênio , Microambiente TumoralRESUMO
Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.
Assuntos
Caseína Quinase Idelta/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Sequência de Aminoácidos , Caseína Quinase Idelta/genética , Hipóxia Celular/fisiologia , Células HEK293 , Células HeLa , Humanos , Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Vitamin D is a hormone that, through its action, elicits a broad spectrum of physiological responses ranging from classic to nonclassical actions such as bone morphogenesis and immune function. In parallel, many studies describe the antiproliferative, proapoptotic, antiangiogenic effects of calcitriol (the active hormonal form) that contribute to its anticancer activity. Additionally, epidemiological data signify the inverse correlation between vitamin D levels and cancer risk. On the contrary, tumors possess several adaptive mechanisms that enable them to evade the anticancer effects of calcitriol. Such maladaptive processes are often a characteristic of the cancer microenvironment, which in solid tumors is frequently hypoxic and elicits the overexpression of Hypoxia-Inducible Factors (HIFs). HIF-mediated signaling not only contributes to cancer cell survival and proliferation but also confers resistance to anticancer agents. Taking into consideration that calcitriol intertwines with signaling events elicited by the hypoxic status cells, this review examines their interplay in cellular signaling to give the opportunity to better understand their relationship in cancer development and their prospect for the treatment of cancer.
RESUMO
Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases' partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.
Assuntos
Proteínas Serina-Treonina Quinases , Splicing de RNA , Proteínas Serina-Treonina Quinases/metabolismo , Fosforilação , Núcleo Celular/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Proteínas Associadas à Matriz Nuclear , Humanos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Matriz Nuclear/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , RNA Mensageiro/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia Celular/genética , Proteínas Serina-Treonina QuinasesRESUMO
Hypoxia-Inducible Factor 1 (HIF-1), a transcriptional activator, is highly involved in the pathology of cancer. Inhibition of HIF-1 retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to prepare a Molecularly Imprinted Polymer (MIP), which was subsequently used for the selective isolation of new inhibitors from frankincense, a gum resin used as anticancer remedy in traditional medicine. The frankincense components isolated by Solid Phase Extraction on MIP (MIP-SPE), efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. The selective retention of acetyl 11-ketoboswellic acid (AKBA, one of the main bioactive components of frankincense) by MIP led to the revealing of its inhibitory activity on the HIF-1 signaling pathway. AKBA was selectively retained by SPE on the quercetin imprinted polymer, with an imprinting effect of 8.1 ± 4.6. Overall, this study demonstrates the potential of MIP application in the screening, recognition and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Boswellia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Impressão Molecular/métodos , Polímeros/química , Quercetina/química , Cromatografia Líquida de Alta Pressão , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Extração em Fase Sólida , Transcrição Gênica/efeitos dos fármacos , Triterpenos/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
Reduced oxygen availability (hypoxia) is a characteristic of many disorders including cancer. Central components of the systemic and cellular response to hypoxia are the Hypoxia Inducible Factors (HIFs), a small family of heterodimeric transcription factors that directly or indirectly regulate the expression of hundreds of genes, the products of which mediate adaptive changes in processes that include metabolism, erythropoiesis, and angiogenesis. The overexpression of HIFs has been linked to the pathogenesis and progression of cancer. Moreover, evidence from cellular and animal models have convincingly shown that targeting HIFs represents a valid approach to treat hypoxia-related disorders. However, targeting transcription factors with small molecules is a very demanding task and development of HIF inhibitors with specificity and therapeutic potential has largely remained an unattainable challenge. Another promising approach to inhibit HIFs is to use peptides modelled after HIF subunit domains known to be involved in protein-protein interactions that are critical for HIF function. Introduction of these peptides into cells can inhibit, through competition, the activity of endogenous HIFs in a sequence and, therefore also isoform, specific manner. This review summarizes the involvement of HIFs in cancer and the approaches for targeting them, with a special focus on the development of peptide HIF inhibitors and their prospects as highly-specific pharmacological agents.
RESUMO
The hypoxia-inducible factor HIF-1 is essential for oxygen homeostasis. Despite its well-understood oxygen-dependent expression, regulation of its transcriptional activity remains unclear. We show that phosphorylation by extracellular signal-regulated kinases1/2 (ERK1/2), in addition to promoting HIF-1α nuclear accumulation, also enhances its interaction with chromatin and stimulates direct binding to nucleophosmin (NPM1), a histone chaperone and chromatin remodeler. NPM1 is required for phosphorylation-dependent recruitment of HIF-1 to hypoxia response elements, its interaction with acetylated histones, and high expression of HIF-1 target genes under hypoxia. Transcriptome analysis revealed a significant number of hypoxia-related genes commonly regulated by NPM1 and HIF-1. These NPM1/HIF-1α co-upregulated genes are enriched in three different cancer types, and their expression correlates with hypoxic tumor status and worse patient prognosis. In concert, silencing of NPM1 expression or disruption of its association with HIF-1α inhibits metabolic adaptation of cancer cells and triggers apoptotic death upon hypoxia. We suggest that ERK-mediated phosphorylation of HIF-1α regulates its physical interaction with NPM1, which is essential for the productive association of HIF-1 with hypoxia target genes and their optimal transcriptional activation, required for survival under low oxygen or tumor growth.
Assuntos
Cromatina , Neoplasias , Hipóxia Celular/genética , Histonas/metabolismo , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/genética , Nucleofosmina , Transdução de SinaisRESUMO
Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1alpha subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1alpha as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC(50)=5.16microM). The mechanism of this inhibition did not involve suppression of HIF-1alpha protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC(50)=4.75microM). Exposure of Huh7 cells to 10microM kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10microM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.