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1.
Acta Biochim Biophys Sin (Shanghai) ; 54(5): 708-715, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35920196

RESUMO

The oncoprotein transcription factor paired box 2 (PAX2) is aberrantly expressed in cancers, but the underlying mechanism remains elusive. Prolyl hydroxylase 3 (PHD3) hydroxylates the proline residue of HIFα, mediating HIFα degradation. The von Hippel-Lindau protein (pVHL) is an E3 ligase which mediates ubiquitination and degradation of hydroxylated HIFα. PHD3 and pVHL are found to inhibit the expression of PAX2, however, the molecular mechanism is unclear. Here we demonstrate that PHD3 hydroxylates PAX2 at proline 9, which is required for pVHL to mediate PAX2 ubiquitination and degradation. Overexpression of PHD3 enhances prolyl hydroxylation, ubiquitination and degradation of PAX2 with little effect on those of PAX2(P9A). PHD3 does not influence PAX2 expression in VHL-null cells. pVHL binds to PAX2 and enhances PAX2 ubiquitination and degradation. However, pVHL does not bind with PAX2(P9A) and cannot enhance its ubiquitination and degradation. Our results suggest that proline 9 hydroxylation is a prerequisite for PAX2 degradation by pVHL. Functional studies indicate that introduction of PAX2 into PAX2-null COS-7 cells promotes cell proliferation, which is suppressed by co-expression of PHD3 but not by hydroxylase-deficient PHD3(H196A). PHD3 inhibits PAX2-induced, but not PAX2(P9A)-induced proliferation of COS-7 cells. These results suggest that PHD3 hydroxylates PAX2, followed by pVHL-mediated PAX2 ubiquitination and degradation. This study also suggests that PHD3 inhibits cell proliferation through downregulating PAX2.


Assuntos
Prolina , Prolil Hidroxilases , Proliferação de Células , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase , Ubiquitina-Proteína Ligases/metabolismo
2.
J Am Coll Surg ; 235(2): 240-254, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35758926

RESUMO

BACKGROUND: Our earlier studies showed that inhibiting prolyl-4-hydroxylase enzymes (PHD-1 and PHD-3) improves angiogenesis, heart function, and limb perfusion in mouse models via stabilizing hypoxia-inducible transcription factor-alpha (HIF-1α). The present study explored the effects of the prolyl-4-hydroxylase enzyme, PHD-2, on ischemic heart failure using cardiac-specific PHD-2 gene knockout (KO) mice (PHD2 -/- ). STUDY DESIGN: Adult wild-type (WT) and PHD2 -/- mice, 8-12 weeks old, were subjected to myocardial infarction (MI) by irreversibly ligating the left anterior descending (LAD) coronary artery. All sham group mice underwent surgery without LAD ligation. Animals were divided into 4 groups: (1) wild-type sham (WTS); (2) wild-type myocardial infarction (WTMI); (3) PHD2KO sham (PHD2 -/- S); (4) PHD2KO myocardial infarction (PHD2 -/- MI). Left ventricular tissue samples collected at various time points after surgery were used for microRNA expression profiling, Western blotting, and immunohistochemical analysis. RESULTS: Volcano plot analysis revealed 19 differentially-expressed miRNAs in the PHD2 -/- MI group compared with the WTMI group. Target analysis using Ingenuity Pathway Analysis showed several differentially regulated miRNAs targeting key signaling pathways such as Akt, VEGF, Ang-1, PTEN, apoptosis, and hypoxia pathways. Western blot analysis showed increased HIF-1α, VEGF, phospho-AKT, ß-catenin expression and reduced Bax expression for the PHD2 -/- MI group compared with the WTMI group. Echocardiographic analysis showed preserved heart functions, and picrosirius red staining revealed decreased fibrosis in PHD2 -/- MI compared with the WTMI group. CONCLUSIONS: PHD2 inhibition showed preserved heart function, enhanced angiogenic factor expression, and decreased apoptotic markers after MI. Overall, cardiac PHD2 gene inhibition is a promising candidate for managing cardiovascular diseases.


Assuntos
MicroRNAs , Infarto do Miocárdio , Animais , Modelos Animais de Doenças , Hipóxia , Isquemia , Camundongos , Miócitos Cardíacos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular
3.
Oncogene ; 41(29): 3665-3679, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35705735

RESUMO

EGLN1, EGLN2 and EGLN3 are proline hydroxylase whose main function is the regulation of the HIF factors. They work as oxygen sensors and are the main responsible of HIFα subunits degradation in normoxia. Being their activity strictly oxygen-dependent, when oxygen tension lowers, their control on HIFα is released, leading to activation of systemic and cellular response to hypoxia. However, EGLN family members activity is not limited to HIF modulation, but it includes the regulation of essential mechanisms for cell survival, cell cycle metabolism, proliferation and transcription. This is due to their reported hydroxylase activity on a number of non-HIF targets and sometimes to hydroxylase-independent functions. For these reasons, EGLN enzymes appear fundamental for development and progression of different cancer types, playing either a tumor-suppressive or a tumor-promoting role, according to EGLN isoform and to tumor context. Notably, EGLN1, the most studied isoform, has been shown to have also a central role in tumor micro-environment modulation, mediating CAF activation and impairing HIF1α -related angiogenesis, thus covering an important function in cancer metastasis promotion. Considering the recent knowledge acquired on EGLNs, the possibility to target these enzymes for cancer treatment is emerging. However, due to their multifaceted and controversial roles in different cancer types, the use of EGLN inhibitors as anti-cancer drugs should be carefully evaluated in each context.


Assuntos
Neoplasias , Prolil Hidroxilases , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Microambiente Tumoral
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(5): 665-672, 2022 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-35673909

RESUMO

OBJECTIVE: To investigate the role of proline 4-hydroxylase Ⅱ (P4HA2) in the occurrence and progression of liver cancer. METHODS: GEPIA and Human Protein Atlas database were used to predict the expression of P4HA2 in hepatocellular carcinoma (HCC), and K-M plotter online database was used to analyze the relationship between P4HA2 expression and the prognosis of HCC. We also examined the expressions of P4HA2 in HCC cells and normal hepatocytes using qRT-PCR and Western blotting. With lentivirus-mediated RNA interference, P4HA2 expression was knocked down in hepatoma SNU-449 and Hep-3B cells, and the changes in cell proliferation, migration and invasion were assessed using cell counting kit-8 (CCK-8) assay, colony formation test, scratch test and Transwell assay. The changes in the expressions of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR signal pathway-related proteins were detected using Western blotting. RESULTS: Online database analysis showed that the expression of P4HA2 was significantly higher in HCC tissues than in normal liver tissues (P < 0.05). The expression levels of P4HA2 mRNA and protein were also significantly higher in HCC cell lines than in normal hepatocytes (P < 0.01). Lentivirus-mediated RNA interference of P4HA2 significantly lowered the expression levels of P4HA2 mRNA and protein in the hepatoma cells (P < 0.05) and caused obvious inhibition of cell proliferation, migration and invasion. P4HA2 knockdown significantly increased the expression of E-cadherin protein, lowered the expressions of N-cadherin and Snail, and obviously decreased the expressions of phosphorylated PI3K, AKT and mTOR (P < 0.05). CONCLUSION: P4HA2 enhances the proliferation, migration, invasion, and EMT of hepatoma cells by activating the PI3K/Akt/mTOR signaling pathway to promote the occurrence and progression of liver cancer.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Prolil Hidroxilases/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Neoplasias Hepáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
5.
Acta Physiol (Oxf) ; 235(4): e13826, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35491502

RESUMO

AIM: Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. METHODS: We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat.  Lectin absorption chromatography was used to assess liver-derived EPO.  In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. RESULTS: In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. CONCLUSIONS: Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.


Assuntos
Eritropoetina , Insuficiência Renal Crônica , Animais , Transdiferenciação Celular , Eritropoetina/farmacologia , Fibrose , Prolina Dioxigenases do Fator Induzível por Hipóxia , Rim , Camundongos , Camundongos Knockout , Miofibroblastos , Néfrons , Prolil Hidroxilases , Insuficiência Renal Crônica/complicações
6.
Am J Physiol Renal Physiol ; 323(1): F81-F91, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35499237

RESUMO

Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Activation of the oxygen-regulated hypoxia-inducible factor (HIF) pathway has been shown to protect mucosal membranes by increasing the expression of cytoprotective genes and by suppressing inflammation. The activity of HIF is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which have been exploited as therapeutic targets for the treatment of anemia of chronic kidney disease. Here, we established a mouse model of acute cyclophosphamide (CYP)-induced blood-urine barrier disruption associated with inflammation and severe urinary dysfunction to investigate the HIF-PHD axis in inflammatory bladder injury. We found that systemic administration of dimethyloxalylglycine or molidustat, two small-molecule inhibitors of HIF-prolyl hydroxylases, profoundly mitigated CYP-induced bladder injury and inflammation as assessed by morphological analysis of transmural edema and urothelial integrity and by measuring tissue cytokine expression. Void spot analysis to examine bladder function quantitatively demonstrated that HIF-prolyl hydroxylase inhibitor administration normalized micturition patterns and protected against CYP-induced alteration of urinary frequency and micturition patterns. Our study highlights the therapeutic potential of HIF-activating small-molecule compounds for the prevention or therapy of bladder injury and urinary dysfunction due to blood-urine barrier disruption.NEW & NOTEWORTHY Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Here, we demonstrate that pharmacological inhibition of hypoxia-inducible factor (HIF)-prolyl hydroxylation prevented bladder injury and protected from urinary dysfunction in a mouse model of cyclophosphamide-induced disruption of the blood-urine barrier. Our study highlights a potential role for HIF-activating small-molecule compounds in the prevention or therapy of bladder injury and urinary dysfunction and provides a rationale for future clinical studies.


Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia , Bexiga Urinária , Animais , Ciclofosfamida/toxicidade , Modelos Animais de Doenças , Hidroxilação , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Inflamação/metabolismo , Camundongos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/metabolismo , Bexiga Urinária/metabolismo
7.
Int J Mol Sci ; 23(9)2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35563465

RESUMO

We propose a novel mechanism by which cancer cells can modulate the oxygen concentration within the nucleus, potentially creating low nuclear oxygen conditions without the need of an hypoxic micro-environment and suited for allowing cancer cells to resist chemo- and radio-therapy. The cells ability to alter intra-cellular oxygen conditions depends on the amount of cholesterol present within the cellular membranes, where high levels of cholesterol can yield rigid membranes that slow oxygen diffusion. The proposed mechanism centers on the competition between (1) the diffusion of oxygen within the cell and across cellular membranes that replenishes any consumed oxygen and (2) the consumption of oxygen in the mitochondria, peroxisomes, endoplasmic reticulum (ER), etc. The novelty of our work centers around the assumption that the cholesterol content of a membrane can affect the oxygen diffusion across the membrane, reducing the cell ability to replenish the oxygen consumed within the cell. For these conditions, the effective diffusion rate of oxygen becomes of the same order as the oxygen consumption rate, allowing the cell to reduce the oxygen concentration of the nucleus, with implications to the Warburg Effect. The cellular and nucleus oxygen content is indirectly evaluated experimentally for bladder (T24) cancer cells and during the cell cycle, where the cells are initially synchronized using hydroxeaurea (HU) at the late G1-phase/early S-phase. The analysis of cellular and nucleus oxygen concentration during cell cycle is performed via (i) RT-qPCR gene analysis of hypoxia inducible transcription factors (HIF) and prolyl hydroxylases (PHD) and (ii) radiation clonogenic assay every 2 h, after release from synchronization. The HIF/PHD genes allowed us to correlate cellular oxygen with oxygen concentration in the nucleus that is obtained from the cells radiation response, where the amount DNA damage due to radiation is directly related to the amount of oxygen present in the nucleus. We demonstrate that during the S-phase cells can become hypoxic in the late S-phase/early G2-phase and therefore the radiation resistance increases 2- to 3-fold.


Assuntos
Núcleo Celular , Colesterol , Hipóxia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Núcleo Celular/metabolismo , Colesterol/metabolismo , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/metabolismo , Prolil Hidroxilases/metabolismo , Tolerância a Radiação/fisiologia , Fase S
8.
Biotechnol J ; 17(7): e2100698, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35427441

RESUMO

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.


Assuntos
Sistemas CRISPR-Cas , Tabaco , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma de Planta , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , RNA Guia/metabolismo , Tabaco/genética , Tabaco/metabolismo
9.
Genes (Basel) ; 13(3)2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35327962

RESUMO

Osteogenesis imperfecta (OI) is a syndromic disorder of bone fragility with high variation in its clinical presentation. Equally variable is molecular aetiology; recessive forms are caused by approximately 20 different genes, many of which are directly implicated in collagen type I biosynthesis. Biallelic variants in prolyl 3-hydroxylase 1 (P3H1) are known to cause severe OI by affecting the competence of the prolyl 3-hydroxylation-cartilage associated protein-peptidyl-prolyl cis-trans isomerase B (P3H1-CRTAP-CyPB) complex, which acts on the Pro986 residue of collagen type I α 1 (COL1A1) and Pro707 collagen type I α 2 (COL1A2) chains. The investigation of an OI cohort of 146 patients in Vietnam identified 14 families with P3H1 variants. The c.1170+5G>C variant was found to be very prevalent (12/14) and accounted for 10.3% of the Vietnamese OI cohort. New P3H1 variants were also identified in this population. Interestingly, the c.1170+5G>C variants were found in families with the severe clinical Sillence types 2 and 3 but also the milder types 1 and 4. This is the first time that OI type 1 is reported in patients with P3H1 variants expanding the clinical spectrum. Patients with a homozygous c.1170+5G>C variant shared severe progressively deforming OI type 3: bowed long bones, deformities of ribcage, long phalanges and hands, bluish sclera, brachycephaly, and early intrauterine fractures. Although it remains unclear if the c.1170+5G>C variant constitutes a founder mutation in the Vietnamese population, its prevalence makes it valuable for the molecular diagnosis of OI in patients of the Kinh ethnicity. Our study provides insight into the clinical and genetic variation of P3H1-related OI in the Vietnamese population.


Assuntos
Glicoproteínas de Membrana/genética , Osteogênese Imperfeita , Prolil Hidroxilases/genética , Proteoglicanas/genética , Asiáticos , Variação Biológica da População , Colágeno Tipo I/genética , Proteínas da Matriz Extracelular/genética , Humanos , Chaperonas Moleculares/genética , Mutação , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Vietnã/epidemiologia
11.
J Biol Chem ; 298(3): 101721, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35151685

RESUMO

Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm-/- mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm-/- mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.


Assuntos
Barreira Hematoencefálica , Infarto da Artéria Cerebral Média , Prolil Hidroxilases , Acidente Vascular Cerebral , Animais , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/metabolismo , Permeabilidade da Membrana Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/metabolismo , Camundongos , /metabolismo , Permeabilidade , Prolil Hidroxilases/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Elife ; 112022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35192456

RESUMO

The oxygen sensor prolyl hydroxylase domain 2 (PHD2) plays an important role in cell hypoxia adaptation by regulating the stability of HIF proteins (HIF1α and HIF2α) in numerous cell types, including T lymphocytes. The role of oxygen sensor on immune cells, particularly on regulatory T cell (Treg) function, has not been fully elucidated. The purpose of our study was to evaluate the role of PHD2 in the regulation of Treg phenotype and function. We demonstrate herein that selective ablation of PHD2 expression in Treg (PHD2ΔTreg mice) leads to a spontaneous systemic inflammatory syndrome, as evidenced by weight loss, development of a rectal prolapse, splenomegaly, shortening of the colon, and elevated expression of IFN-γ in the mesenteric lymph nodes, intestine, and spleen. PHD2 deficiency in Tregs led to an increased number of activated CD4 conventional T cells expressing a Th1-like effector phenotype. Concomitantly, the expression of innate-type cytokines such as Il1b, Il12a, Il12b, and Tnfa was found to be elevated in peripheral (gut) tissues and spleen. PHD2ΔTreg mice also displayed an enhanced sensitivity to dextran sodium sulfate-induced colitis and toxoplasmosis, suggesting that PHD2-deficient Tregs did not efficiently control inflammatory response in vivo, particularly those characterized by IFN-γ production. Further analysis revealed that Treg dysregulation was largely prevented in PHD2-HIF2α (PHD2-HIF2αΔTreg mice), but not in PHD2-HIF1α (PHD2-HIF1αΔTreg mice) double KOs, suggesting an important and possibly selective role of the PHD2-HIF2α axis in the control of Treg function. Finally, the transcriptomic analysis of PHD2-deficient Tregs identified the STAT1 pathway as a target of the PHD2-HIF2α axis in regulatory T cell phenotype and in vivo function.


Assuntos
Colite , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Linfócitos T Reguladores , Animais , Colite/induzido quimicamente , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Oxigênio , Pró-Colágeno-Prolina Dioxigenase , Prolil Hidroxilases
13.
Sci Rep ; 12(1): 2928, 2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35190588

RESUMO

Biomarkers for treatment sensitivity or drug resistance used in precision medicine include prognostic and predictive molecules, critical factors in selecting appropriate treatment protocols and improving survival rates. However, identification of accurate biomarkers remain challenging due to the high risk of false-positive findings and lack of functional validation results for each biomarker. Here, we discovered a mechanical correlation between leucine proline-enriched proteoglycan 1 (LEPRE1) and pelitinib drug sensitivity using in silico statistical methods and confirmed the correlation in acute myeloid leukemia (AML) and A549 lung cancer cells. We determined that high LEPRE1 levels induce protein kinase B activation, overexpression of ATP-binding cassette superfamily G member 2 (ABCG2) and E-cadherin, and cell colonization, resulting in a cancer stem cell-like phenotype. Sensitivity to pelitinib increases in LEPRE1-overexpressing cells due to the reversing effect of ABCG2 upregulation. LEPRE1 silencing induces pelitinib resistance and promotes epithelial-to-mesenchymal transition through actin rearrangement via a series of Src/ERK/cofilin cascades. The in silico results identified a mechanistic relationship between LEPRE1 and pelitinib drug sensitivity, confirmed in two cancer types. This study demonstrates the potential of LEPRE1 as a biomarker in cancer through in-silico prediction and in vitro experiments supporting the clinical development of personalized medicine strategies based on bioinformatics findings.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminoquinolinas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Transição Epitelial-Mesenquimal/genética , Regulação Leucêmica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Prolil Hidroxilases/genética , Prolil Hidroxilases/fisiologia , Proteoglicanas/genética , Proteoglicanas/fisiologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Neoplasias Pulmonares/diagnóstico
14.
Cells ; 11(4)2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35203399

RESUMO

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.


Assuntos
Anemia , Eritropoetina , Anemia/metabolismo , Animais , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Glicina/análogos & derivados , Hipóxia/metabolismo , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Rim/metabolismo , Camundongos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/metabolismo
15.
Protein Expr Purif ; 194: 106073, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35192944

RESUMO

Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates the hypoxia inducible factor (HIF) which then targets it for degradation in oxygenated cells. Inhibition of the activity of the PHD2 enzyme under hypoxic environmental conditions acts to upregulate HIF. Thus, PHD2 inhibitors may serve as a promising treatment for HIF-dependent diseases. In this study, recombinant PHD2 protein was successfully expressed using a baculovirus-insect cell expression secretory system. PHD2 was purified and in combination with bacterially expressed functional von Hippel Lindau protein-elongin B-elongin C (VBC) protein complex was used to successfully develop a fluorescence-based PHD2 activity assay. Myricetin was identified as a novel potent PHD2 inhibitor by high-throughput screening of a natural compound library. Further studies showed that treatment of human neuroblastoma SH-SY5Y cells with Myricetin increased HIF-1α protein levels. These results indicate that the insect cell expression system is capable of producing highly active recombinant PHD2 protein from which a fluorescence-based activity assay can be developed for high-throughput screening applications.


Assuntos
Dioxigenases , Prolina Dioxigenases do Fator Induzível por Hipóxia , Animais , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Insetos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
16.
J Immunol ; 208(5): 1280-1291, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35121641

RESUMO

Inflammatory bowel disease such as chronic colitis promotes colorectal cancer, which is a common cause of cancer mortality worldwide. Hypoxia is a characteristic of inflammation as well as of solid tumors and enforces a gene expression response controlled by hypoxia-inducible factors (HIFs). Once established, solid tumors are immunosuppressive to escape their abatement through immune cells. Although HIF activity is known to 1) promote cancer development and 2) drive tumor immune suppression through the secretion of adenosine, both prolyl hydroxylases and an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) negatively regulate HIF. Thus, FIH may act as a tumor suppressor in colorectal cancer development. In this study, we examined the role of colon epithelial FIH in a mouse model of colitis-induced colorectal cancer. We recapitulated colitis-associated colorectal cancer development in mice using the azoxymethane/dextran sodium sulfate model in Vil1-Cre/FIH+f/+f and wild-type siblings. Colon samples were analyzed regarding RNA and protein expression and histology. Vil1-Cre/FIH+f/+f mice showed a less severe colitis progress compared with FIH+f/+f animals and a lower number of infiltrating macrophages in the inflamed tissue. RNA sequencing analyses of colon tissue revealed a lower expression of genes associated with the immune response in Vil1-Cre/FIH+f/+f mice. However, tumor occurrence did not significantly differ between Vil1-Cre/FIH+f/+f and wild-type mice. Thus, FIH knockout in colon epithelial cells did not modulate colorectal cancer development but reduced the inflammatory response in chronic colitis.


Assuntos
Neoplasias Associadas a Colite/patologia , Colite/patologia , Neoplasias Colorretais/patologia , Mucosa Intestinal/patologia , Oxigenases de Função Mista/metabolismo , Adenosina/metabolismo , Animais , Azoximetano/toxicidade , Hipóxia Celular/fisiologia , Colite/induzido quimicamente , Colite/genética , Neoplasias Associadas a Colite/genética , Colo/patologia , Neoplasias Colorretais/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigenases de Função Mista/genética , Prolil Hidroxilases/metabolismo , Transdução de Sinais/fisiologia , Evasão Tumoral/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
17.
Genes (Basel) ; 13(1)2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35052464

RESUMO

Osteogenesis imperfecta (OI) is an inherited disease of bone characterized by increased bone fragility. Here, we report the results of the molecular architecture of osteogenesis imperfecta research in patients from Bashkortostan Republic, Russia. In total, 16 mutations in COL1A1, 11 mutations in COL1A2, and 1 mutation in P3H1 and IFIMT5 genes were found in isolated states; 11 of them were not previously reported in literature. We found mutations in CLCN7, ALOX12B, PLEKHM1, ERCC4, ARSB, PTH1R, and TGFB1 that were not associated with OI pathogenesis in patients with increased bone fragility. Additionally, we found combined mutations (c.2869C>T, p. Gln957* in COL1A1 and c.1197+5G>A in COL1A2; c.579delT, p. Gly194fs in COL1A1 and c.1197+5G>A in COL1A2; c.2971G>C, p. Gly991Arg in COL1A2 and c.212G>C, p.Ser71Thr in FGF23; c.-14C>T in IFITM5 and c.1903C>T, p. Arg635* in LAMB3) in 4 patients with typical OI clinic phenotypes.


Assuntos
Colágeno Tipo I/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação , Osteogênese Imperfeita/patologia , Prolil Hidroxilases/genética , Proteoglicanas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese Imperfeita/epidemiologia , Osteogênese Imperfeita/genética , Fenótipo , Federação Russa , Adulto Jovem
18.
Acta Haematol ; 145(4): 412-418, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35051929

RESUMO

INTRODUCTION: Hypoxia-inducible factor prolyl hydroxylase domain inhibitors (HIF-PHI) are a new treatment for renal anemia. HIF-PHI is believed to increase iron usage to improve availability of iron for erythropoiesis. Therefore, there is concern that HIF-PHI might be prone to iron deficiency and that thrombosis might be induced by increased platelet and transferrin levels due to this iron deficiency. METHODS: Relationship of iron-related factors with platelet count (PLT) and total iron-binding capacity (TIBC; which reflects the transferrin level) were examined in 29 patients who were treated with darbepoetin alfa (DA) and then switched to roxadustat (Rox). To determine how changes in PLT and TIBC related to changes in iron-related factors, univariable and multivariable linear regression models were applied. To examine what iron-related factors on Day 0 influenced change in PLT, we used receiver operating characteristic (ROC) curves and logistic regression analysis for a rate of change in PLT ≤0% as the endpoint. Logistic regression analysis was performed with the reference group having serum ferritin (s-ft) or Transferrin saturation below the corresponding cutoff value (low vs. high). RESULTS: Multivariable analysis showed significant positive correlations between the rate of change in PLT and the change in s-ft and red blood cells (RBC) count {ß-coefficients; 0.40 [95% confidence interval (CI): 0.17-0.62], p = 0.001} (ß-coefficients; 30.45 [95% CI: 10.90-50.00], p = 0.004). The rate of change in TIBC was significantly positively correlated with only the change in RBC count. The ROC showed a significant cutoff value for s-ft of 77.2 ng/mL (sensitivity 63.6%, specificity 83.3%, area under the curve 0.76, 95% CI 0.55-0.96). Multivariable logistic regression also showed that only high s-ft was significantly elevated (9.46, 95% CI 1.42-63.30, p = 0.020). CONCLUSIONS: This study showed that changes in PLT were correlated with s-ft and amount of hematopoiesis. This suggests that an increase in PLT due to iron levels is less likely when s-ft is 77.2 ng/mL or higher at the time of switching from DA to Rox. In contrast, TIBC was only related to hematopoiesis in these patients. Control of s-ft before initiation of HIF-PHI treatment and gradual hematopoiesis might reduce the risk of thrombosis when switching from erythropoiesis-stimulating agents to HIF-PHI.


Assuntos
Inibidores de Prolil-Hidrolase , Insuficiência Renal Crônica , Darbepoetina alfa , Ferritinas , Humanos , Hipóxia , Ferro , Prolil Hidroxilases , Inibidores de Prolil-Hidrolase/uso terapêutico , Insuficiência Renal Crônica/terapia , Transferrinas
19.
Angiogenesis ; 25(2): 259-274, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34997404

RESUMO

Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved  actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.


Assuntos
Hipertensão Pulmonar , Animais , Artérias/metabolismo , Células Endoteliais/metabolismo , Fibrose , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos , Miócitos de Músculo Liso/patologia , Prolil Hidroxilases/metabolismo
20.
Mitochondrion ; 63: 51-56, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35066139

RESUMO

Iron deficiency is observed in nearly half of the heart failure patients whilst closely correlated with mitochondrial dysfunction. Besides the structural roles in mitochondria, iron is also the cofactor of the hypoxia inducible factor (Hif) degradating enzyme, prolylhydroxylase, thereby Hif accumulation and its related metabolic effects commonly involve in iron deficiency. In this study, we used atrium derived HL-1 cells to investigate the effects of iron depletion on mitochondrial function under in vitro conditions. We aimed to discriminate the Hif dependent effects of iron deprivation on mitochondrial function to reveal the mechanisms leading to cardiac failure. For this purpose, HL-1 cells were either directly incubated with the iron chelating agent deferoxamine (DFO) or with dimethyloxalylglycine (DMOG, inhibitor of prolylhydroxylase). Mitochondrial function was evaluated by measuring cellular ATP content and mitochondrial potential (Ψ). According to our results, 48 h of DFO incubation affected cell viability and ATP production through further mechanisms additional to Hif-1α accumulation. Unlike DMOG group, DFO incubation did not disturb mitochondrial function probably due to its low permeability. Whether or not, prolyl hydroxylase inhibition without iron depletion may negatively affect mitochondrial function through Hif dependent mechanisms.


Assuntos
Prolil Hidroxilases , Trifosfato de Adenosina , Humanos , Ferro/metabolismo , Mitocôndrias/metabolismo
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