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1.
Cell ; 149(1): 63-74, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22464323

RESUMO

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.


Assuntos
Eritropoese , Eritropoetina/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Anemia/prevenção & controle , Animais , Células Precursoras Eritroides/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/metabolismo , Camundongos , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(16): e2210418120, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37040401

RESUMO

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Humanos , Proteínas Culina/metabolismo , Hipóxia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Genes abl , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo
3.
Genes Dev ; 29(8): 817-31, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25846796

RESUMO

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.


Assuntos
Osso e Ossos/enzimologia , Homeostase , Osteoprotegerina/metabolismo , Oxigênio/metabolismo , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Células 3T3 , Animais , Reabsorção Óssea/genética , Osso e Ossos/citologia , Comunicação Celular , Hipóxia Celular/fisiologia , Células Cultivadas , Ativação Enzimática , Feminino , Inativação Gênica , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais/genética , Células-Tronco/enzimologia
5.
Curr Osteoporos Rep ; 12(4): 428-32, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25204993

RESUMO

Osteoblasts are an important cellular component of the bone microenvironment controlling bone formation and hematopoiesis. Understanding the cellular and molecular mechanisms by which osteoblasts regulate these processes is a rapidly growing area of research given the important implications for bone therapy, regenerative medicine, and hematopoietic stem cell transplantation. Here we summarize our current knowledge regarding the cellular and molecular crosstalk driving bone formation and hematopoiesis and will discuss the implications of a recent finding demonstrating that osteoblasts are a cellular source of erythropoietin .


Assuntos
Eritropoetina/metabolismo , Osteoblastos/metabolismo , Animais , Hematopoese/fisiologia , Humanos , Modelos Animais , Osteoblastos/citologia , Osteogênese/fisiologia , Transdução de Sinais/fisiologia
6.
Genet Med ; 15(3): 229-33, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23037933

RESUMO

PURPOSE: We provide background information/education for national recommendations to include initial newborn screening dried bloodspot serial numbers in electronic birth registrations. Mutual data linking would provide quality checks for each data source, determinations of percentages of newborns screened, and identification of locations where screening is lacking. METHODS: State newborn screening dried bloodspot programs were surveyed to determine the extent of newborn screening dried bloodspot and electronic birth registration linking and the states' level of interest in such linkages. These data were reviewed with federal and state policy makers and presented to the Secretary of Health and Human Services' Advisory Committee on Heritable Disorders in Newborns and Children for national policy recommendations. RESULTS: Only 40% of state newborn screening dried bloodspot programs reported comparing births with screens. All states use serially numbered newborn screening dried bloodspot collection cards, and electronic birth registrations exist in almost all states. Newborn screening dried bloodspot serial number data fields currently exist in only 24% of state electronic birth registrations. CONCLUSION: The Secretary of Health and Human Services' Advisory Committee on Heritable Disorders in Newborns and Children recommends the universal use of the newborn screening dried bloodspot serial number in a standardized format as part of state birth registration; consideration of including the initial newborn screening dried bloodspot serial number as a required data field; and, once established, using these data linkages to monitor completeness of newborn screening and to validate demographic information in both systems.


Assuntos
Declaração de Nascimento/legislação & jurisprudência , Doenças Genéticas Inatas/diagnóstico , Triagem Neonatal/instrumentação , Triagem Neonatal/normas , Comitês Consultivos , Humanos , Recém-Nascido
7.
J Vis Exp ; (194)2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37154559

RESUMO

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.


Assuntos
Células da Medula Óssea , Células-Tronco Mesenquimais , Camundongos , Animais , Células-Tronco Hematopoéticas , Hematopoese/fisiologia , Oxigênio , Diferenciação Celular , Células Estromais
8.
Sci Transl Med ; 15(724): eabo5217, 2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-38019933

RESUMO

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Doenças Ósseas Metabólicas , Humanos , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Receptores para Leptina , Camundongos Knockout , Células-Tronco , Subunidade alfa do Fator 1 Induzível por Hipóxia
9.
Elife ; 112022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35179487

RESUMO

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.


Assuntos
Medula Óssea , Condrócitos , Adipócitos , Animais , Diferenciação Celular , Camundongos , Osteoblastos , Osteogênese , RNA/metabolismo , Células-Tronco/metabolismo
10.
Methods Mol Biol ; 2230: 345-356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197024

RESUMO

Oxygen serves as a critical environmental factor essential for maintaining the physiological state of a tissue. Hypoxia, or low oxygen, triggers a cascade of events which allows for cells to adapt to low oxygen tensions and to facilitate oxygen delivery required to maintain tissue homeostasis. In the bone microenvironment (BME), vascular heterogeneity, poor perfusion rates of blood vessels, and high metabolic activity of hematopoietic cells result in the generation of a unique hypoxic landscape. Importantly, in this region, hypoxia and its downstream effectors are associated with establishing stem cell niches and regulating the differentiation of committed progenitors. Given the functional importance of the hypoxic bone niche, visualizing regions of hypoxia may provide valuable insights into the mechanisms that regulate tissue homeostasis. Here, we describe the utilization of the nitroimidazole derivative, pimonidazole, to detect hypoxic regions within the BME.


Assuntos
Osso e Ossos/ultraestrutura , Células-Tronco Hematopoéticas/ultraestrutura , Imagem Molecular/métodos , Nitroimidazóis/farmacologia , Animais , Vasos Sanguíneos/ultraestrutura , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Hipóxia Celular/genética , Microambiente Celular/genética , Homeostase/genética , Humanos , Oxigênio/metabolismo , Nicho de Células-Tronco/genética
11.
Stem Cell Res ; 53: 102317, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33848794

RESUMO

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSCs continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation is widely based on adherence to tissue culture plastic; however, high hematopoietic contamination is a significant impediment in murine models. Remarkably, when cultured at a physiological oxygen tension of 1% O2, a 10-fold reduction in CD45+ hematopoietic cells associated with a concomitant increase in PDGFRα+ stromal cells occur. This is due, in part, to a differential response of the two populations to hypoxia. In standard tissue culture conditions of 21% O2, CD45+ cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O2. In contrast, PDGFR α+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR α+ stromal cell population in murine BMSC cultures.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Hipóxia , Camundongos , Células Estromais
12.
J Bone Miner Res ; 35(10): 2004-2014, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32453500

RESUMO

Skeletal stem/progenitor cells (SSPC) are critical regulators of bone homeostasis by providing a continuous supply of osteoblasts throughout life. In response to inductive signals, SSPC proliferate before osteoblast differentiation. Proliferation requires the duplication of all cellular components before cell division. This imposes a unique biosynthetic requirement for amino acids that can be used for biomass production. Thus, the ability to sense and respond to amino acid availability is likely a major determinant for proliferation. Using a cellular and genetic approach, we demonstrate the amino acid sensor GCN2 is required to support the robust proliferative capacity of SSPC during bone homeostasis. GCN2 ablation results in decreased postnatal bone mass due primarily to reduced osteoblast numbers. Decreased osteoblast numbers is likely attributed to reduced SSPC proliferation as loss of GCN2 specifically affected proliferation in cultured bone marrow stromal cells (BMSCs) without impacting osteoblast differentiation in vitro. Mechanistically, GCN2 regulates proliferation by increasing amino acid uptake downstream of the transcriptional effector ATF4. Collectively, these data suggest amino acid sensing through the GCN2/ATF4 pathway is indispensable for robust SSPC proliferation necessary for bone homeostasis. © 2020 American Society for Bone and Mineral Research.


Assuntos
Proliferação de Células , Osteoblastos/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Camundongos
13.
Differentiation ; 76(2): 193-205, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17608733

RESUMO

E-cadherin expression is unusually regulated in epithelial ovarian carcinoma. It is not expressed in poorly cohesive ovarian surface epithelial (OSE) target cells, but is expressed in cohesive pre-malignant lesions and in highly cohesive, well-differentiated tumors where it is membrane associated, presumably in adherens junctions. E-cadherin expression is subsequently suppressed, or its function is disrupted, in late-stage invasive tumors. Here, we observed that increased E-cadherin expression in ovarian carcinoma cells was associated with increased E-cadherin promoter activity, increased adherens junction formation, decreased beta-catenin signaling-dependent LEF-1 activity, and the generation of cohesive spheroids in basement membrane gel culture. Forced expression of wild-type E-cadherin in immortalized OSE cells initiated adherens junction formation, decreased LEF-1 activity, decreased the mesenchymal migration that is a characteristic of OSE cells that have been maintained in monolayer culture, and induced the formation of cohesive spheroids in basement membrane gels. Conversely, forced expression of a dominant-negative E-cadherin mutant in ovarian carcinoma cells disrupted adherens junctions, increased mesenchymal cell migration, and prevented spheroidal morphogenesis without altering LEF-1 signaling. Therefore, in addition to suppressing late-stage tumor progression, E-cadherin-mediated adherens junctions may also contribute to the initial emergence of a cohesive morphogenic phenotype that is a hallmark of differentiated epithelial ovarian carcinoma.


Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Carcinoma/patologia , Neoplasias Ovarianas/patologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/citologia , Feminino , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Neoplasias Ovarianas/metabolismo
14.
Cancer Res ; 67(17): 8216-22, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17804735

RESUMO

Although many cancers are maintained by tumor-initiating cells, this has not been shown for mesenchymal tumors, in part due to the lack of unique surface markers that identify mesenchymal progenitors. An alternative technique to isolate stem-like cells is to isolate side population (SP) cells based on efflux of Hoechst 33342 dye. We examined 29 mesenchymal tumors ranging from benign to high-grade sarcomas and identified SP cells in all but six samples. There was a positive correlation between the percentage of SP cells and the grade of the tumor. SP cells preferentially formed tumors when grafted into immunodeficient mice, and only cells from tumors that developed from the SP cells had the ability to initiate tumor formation upon serial transplantation. Although SP cells are able to efflux rhodamine dye in addition to Hoechst 33342, we found that the ability to efflux rhodamine dye did not identify a population of cells enriched for tumor-initiating capacity. Here, we identify a subpopulation of cells within a broad range of benign and malignant mesenchymal tumors with tumor-initiating capacity. In addition, our data suggest that the proportion of SP cells could be used as a prognostic factor and that therapeutically targeting this subpopulation of cells could be used to improve patient outcome.


Assuntos
Neoplasias de Tecido Conjuntivo e de Tecidos Moles/patologia , Células-Tronco Neoplásicas/citologia , Animais , Proliferação de Células , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias/patologia , Células-Tronco Neoplásicas/transplante , Transplante Heterólogo
15.
Cancer Lett ; 268(1): 1-9, 2008 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-18487012

RESUMO

Cancer stem cells (CSCs) are found in multiple tumor types. While the presence of surface markers selectively expressed on CSCs are used to isolate these cells, no marker or pattern of makers are known to prospectively identify CSCs in many tumor types. In such cases exploitation of stem cell characteristics can be used to identify CSCs and one such characteristic is the capacity to extrude dyes such as Hoechst 33342. Cell that exclude this dye are referred to as side population (SP) cells. These cells share characteristics of CSCs, specifically, they are enriched for tumor initiating capacity, they express stem-like genes, and they are resistant to chemotherapeutic drugs. Dye exclusion is a valuable technique as it identifies a unique population of cells with stem-like characteristics.


Assuntos
Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Previsões , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos
16.
Cell Rep ; 12(1): 116-127, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26119730

RESUMO

Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.


Assuntos
Carcinogênese/metabolismo , Carcinoma de Células Renais/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição/metabolismo , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismo
17.
Sci Transl Med ; 6(236): 236ra64, 2014 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-24828078

RESUMO

Radiation-induced gastrointestinal (GI) toxicity can be a major source of morbidity and mortality after radiation exposure. There is an unmet need for effective preventative or mitigative treatments against the potentially fatal diarrhea and water loss induced by radiation damage to the GI tract. We report that prolyl hydroxylase inhibition by genetic knockout or pharmacologic inhibition of all PHD (prolyl hydroxylase domain) isoforms by the small-molecule dimethyloxallyl glycine (DMOG) increases hypoxia-inducible factor (HIF) expression, improves epithelial integrity, reduces apoptosis, and increases intestinal angiogenesis, all of which are essential for radioprotection. HIF2, but not HIF1, is both necessary and sufficient to prevent radiation-induced GI toxicity and death. Increased vascular endothelial growth factor (VEGF) expression contributes to the protective effects of HIF2, because inhibition of VEGF function reversed the radioprotection and radiomitigation afforded by DMOG. Additionally, mortality from abdominal or total body irradiation was reduced even when DMOG was given 24 hours after exposure. Thus, prolyl hydroxylase inhibition represents a treatment strategy to protect against and mitigate GI toxicity from both therapeutic radiation and potentially lethal radiation exposures.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Lesões por Radiação/tratamento farmacológico , Aminoácidos Dicarboxílicos/química , Animais , Apoptose , Peso Corporal , Linhagem Celular Tumoral , Quelantes/química , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/efeitos da radiação , Regulação da Expressão Gênica , Hematócrito , Heterozigoto , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Inibidores de Prolil-Hidrolase/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Lesões por Radiação/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/química
18.
Nat Med ; 19(10): 1325-30, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24037093

RESUMO

Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout
19.
Cancer Res ; 70(19): 7690-8, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20841474

RESUMO

The cellular origins from which most tumors arise are poorly defined, especially in mesenchymal neoplasms. Aggressive fibromatosis, also known as desmoid tumor, is a locally invasive soft tissue tumor that has mesenchymal characteristics. We found that aggressive fibromatosis tumors express genes and cell surface markers characteristic of mesenchymal stem cells (MSC). In mice that are genetically predisposed to develop aggressive fibromatosis tumors (Apc(wt/1638N)), we found that the number of tumors formed was proportional to the number of MSCs present. Sca-1(-/-) mice, which develop fewer MSCs, were crossed with Apc(wt/1638N) mice. Doubly mutant mice deficient in Sca-1 developed substantially fewer aggressive fibromatosis tumors than wild-type (WT) littermates, but Sca-1 deficiency had no effect on the formation of epithelial-derived intestinal polyps. MSCs isolated from Apc(wt/1638N) mice (or mice expressing a stabilized form of ß-catenin) induced aberrant cellular growth reminiscent of aggressive fibromatosis tumors after engraftment to immunocompromised mice, but WT cells and mature fibroblasts from the same animals did not. Taken together, our findings indicate that aggressive fibromatosis is derived from MSCs, and that ß-catenin supports tumorigenesis by maintaining mesenchymal progenitor cells in a less differentiated state. Protecting this progenitor cell population might prevent tumor formation in patients harboring a germline APC mutation, where fibromatosis is currently the leading cause of mortality.


Assuntos
Fibroma/patologia , Células-Tronco Mesenquimais/patologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Adolescente , Adulto , Alelos , Animais , Criança , Feminino , Fibroma/genética , Fibroma/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Adulto Jovem , beta Catenina/biossíntese
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