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1.
PLoS One ; 18(4): e0284012, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37115796

RESUMO

Cd248 has recently been associated with adipose tissue physiology, demonstrated by reduced weight gain in high fat diet-fed mice with genetic deletion of Cd248 relative to controls. Here we set out to determine the metabolic consequences of loss of Cd248. Strikingly, we find these to be sex specific; By subjecting Cd248-/- and Cd248+/+ mice to a high fat diet and indirect calorimetry study, we identified that only male Cd248-/- mice show reduced weight gain compared to littermate control wildtype mice. In addition, male (but not female) mice showed a lower respiratory exchange ratio on both chow and high fat diets, indicating a predisposition to metabolise lipid. Lipidomic studies on specific fat depots found reduced triglyceride and diglyceride deposition in male Cd248-/- mice, and this was supported by reduced expression of lipogenic and adipogenic genes. Finally, metabolomic analysis of isolated, differentiated preadipocytes found alterations in metabolic pathways associated with lipid deposition in cells isolated from male, but not female, Cd248-/- mice. Overall, our results highlight the importance of sex controls in animal studies and point to a role for Cd248 in sex- and depot-specific regulation of lipid metabolism.


Assuntos
Tecido Adiposo , Lipidômica , Animais , Feminino , Masculino , Camundongos , Tecido Adiposo/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Dieta Hiperlipídica , Metabolismo dos Lipídeos/genética , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismo , Aumento de Peso
2.
Clin Exp Immunol ; 212(1): 1-10, 2023 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-36891817

RESUMO

Dysregulation of leukocyte trafficking, lipid metabolism, and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in lifestyle choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B cells were observed in the secondary lymphoid tissues (e.g. spleen and inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimize the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through lifestyle modifications.


Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Inflamação/patologia , Dieta , Linfócitos T CD4-Positivos/metabolismo , Camundongos Endogâmicos C57BL , Tecido Adiposo
3.
Diabetes ; 72(2): 275-289, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36445949

RESUMO

GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in the transport of circulating vitamin 25(OH)D and fatty acids, as well as actin scavenging. In the pancreatic islets, the gene encoding GC, GC/Gc, is highly localized to glucagon-secreting α-cells. Despite this, the role of GC in α-cell function is poorly understood. We previously showed that GC is essential for α-cell morphology, electrical activity, and glucagon secretion. We now show that loss of GC exacerbates α-cell failure during metabolic stress. High-fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α-cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α-cell GC levels, increase glucagon granule area, and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α-cells of donors with type 2 diabetes, which is associated with changes in α-cell mass, morphology, and glucagon expression. Together, these data demonstrate an important role for GC in α-cell adaptation to metabolic stress.


Assuntos
Diabetes Mellitus Tipo 2 , Globulinas , Animais , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Globulinas/metabolismo , Glucagon/metabolismo , Estresse Fisiológico , Proteína de Ligação a Vitamina D/genética , Proteína de Ligação a Vitamina D/metabolismo
4.
Blood Adv ; 7(1): 46-59, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36269841

RESUMO

Mice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2ß1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2ß1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2ß1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2ß1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.


Assuntos
Plaquetas , Trombocitopenia , Camundongos , Animais , Plaquetas/metabolismo , Megacariócitos/metabolismo , Trombocitopenia/genética , Quinases da Família src/metabolismo , Colágeno/metabolismo
5.
J Endocrinol ; 251(1): 111-123, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34370682

RESUMO

Supplementation with precursors of NAD has been shown to prevent and reverse insulin resistance, mitochondrial dysfunction, and liver damage in mouse models of diet-induced obesity. We asked whether the beneficial effects of supplementation with the NAD precursor nicotinamide riboside (NR) are dependent on mouse strain. We compared the effects of NR supplementation on whole-body energy metabolism and mitochondrial function in mildly obese C57BL/6N and C57BL/6J mice, two commonly used strains to investigate metabolism. Male C57BL/6N and C57BL/6J mice were fed a high-fat diet (HFD) or standard chow with or without NR supplementation for 8 weeks. Body and organ weights, glucose tolerance, and metabolic parameters as well as mitochondrial O2 flux in liver and muscle fibers were assessed. We found that NR supplementation had no influence on body or organ weight, glucose metabolism or hepatic lipid accumulation, energy expenditure, or metabolic flexibility but increased mitochondrial respiration in soleus muscle in both mouse strains. Strain-dependent differences were detected for body and fat depot weight, fasting blood glucose, hepatic lipid accumulation, and energy expenditure. We conclude that, in mild obesity, NR supplementation does not alter metabolic phenotype in two commonly used laboratory mouse strains.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Niacinamida/análogos & derivados , Obesidade/tratamento farmacológico , Compostos de Piridínio/uso terapêutico , Animais , Respiração Celular/efeitos dos fármacos , Dieta Hiperlipídica , Modelos Animais de Doenças , Avaliação de Medicamentos , Intolerância à Glucose/prevenção & controle , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Niacinamida/uso terapêutico , Obesidade/metabolismo
6.
Cell Rep ; 31(11): 107761, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32553153

RESUMO

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.


Assuntos
Comunicação Celular/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Vitamina D/metabolismo , Animais , Transporte Biológico/fisiologia , Secreções Corporais/metabolismo , Humanos , Camundongos Knockout , Fenótipo
7.
Skelet Muscle ; 10(1): 5, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075690

RESUMO

BACKGROUND: Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11ß-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation. METHODS: We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO. RESULTS: H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD+/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability. CONCLUSIONS: These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.


Assuntos
Músculo Esquelético/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Retículo Sarcoplasmático/metabolismo , Acetilcoenzima A/metabolismo , Animais , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Feminino , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Compostos de Piridínio/metabolismo
8.
Blood ; 133(4): 331-343, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30429161

RESUMO

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.


Assuntos
Plaquetas/metabolismo , Integrases/metabolismo , Leucócitos/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Aglutinação , Animais , Células da Medula Óssea/citologia , Proteína Tirosina Quinase CSK , Linhagem da Célula , Tamanho Celular , Marcação de Genes , Homeostase , Contagem de Linfócitos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Fenótipo , Agregação Plaquetária , Fator Plaquetário 4/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Recombinação Genética/genética , Baço/citologia , Quinases da Família src/metabolismo
9.
Blood ; 132(13): 1413-1425, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-29891536

RESUMO

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b-deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F, G6b KO, and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis.


Assuntos
Plaquetas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Trombocitopenia/metabolismo , Animais , Sítios de Ligação , Plaquetas/metabolismo , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Fosforilação , Mutação Puntual , Mapas de Interação de Proteínas , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Receptores Imunológicos/química , Receptores Imunológicos/genética , Transdução de Sinais , Trombocitopenia/genética , Trombocitopenia/patologia , Domínios de Homologia de src
10.
Blood ; 132(13): 1399-1412, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-29898956

RESUMO

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.


Assuntos
Mutação com Perda de Função , Mielofibrose Primária/congênito , Receptores Imunológicos/genética , Trombocitopenia/congênito , Adolescente , Adulto , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Criança , Pré-Escolar , Feminino , Técnicas de Introdução de Genes , Humanos , Lactente , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linhagem , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Trombocitopenia/genética , Trombocitopenia/patologia , Adulto Jovem
11.
Blood ; 131(10): 1122-1144, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29301754

RESUMO

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.


Assuntos
Plaquetas/metabolismo , Homeostase , Trombose/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Animais , Plaquetas/patologia , Proteína Tirosina Quinase CSK , Camundongos , Camundongos Knockout , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Trombose/genética , Quinases da Família src/genética
12.
Blood ; 121(20): 4205-20, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23509158

RESUMO

The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors. In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCγ2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1- and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, αIIbß3, and GPIbα. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function.


Assuntos
Plaquetas/fisiologia , Deleção de Genes , Megacariócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Trombopoese/genética , Animais , Plaquetas/metabolismo , Células Cultivadas , Células Progenitoras de Megacariócitos/metabolismo , Células Progenitoras de Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Trombopoese/fisiologia
13.
Sci Signal ; 5(248): ra78, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23112346

RESUMO

Platelets are highly reactive cell fragments that adhere to exposed extracellular matrix (ECM) and prevent excessive blood loss by forming clots. Paradoxically, megakaryocytes, which produce platelets in the bone marrow, remain relatively refractory to the ECM-rich environment of the bone marrow despite having the same repertoire of receptors as platelets. These include the ITAM (immunoreceptor tyrosine-based activation motif)-containing collagen receptor complex, which consists of glycoprotein VI (GPVI) and the Fc receptor γ-chain, and the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing receptor G6b-B. We showed that mice lacking G6b-B exhibited macrothrombocytopenia (reduced platelet numbers and the presence of enlarged platelets) and a susceptibility to bleeding as a result of aberrant platelet production and function. Platelet numbers were markedly reduced in G6b-B-deficient mice compared to those in wild-type mice because of increased platelet turnover. Furthermore, megakaryocytes in G6b-B-deficient mice showed enhanced metalloproteinase production, which led to increased shedding of cell-surface receptors, including GPVI and GPIbα. In addition, G6b-B-deficient megakaryocytes exhibited reduced integrin-mediated functions and defective formation of proplatelets, the long filamentous projections from which platelets bud off. Together, these findings establish G6b-B as a major inhibitory receptor regulating megakaryocyte activation, function, and platelet production.


Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Receptores Imunológicos/metabolismo , Trombocitopenia/metabolismo , Animais , Plaquetas/patologia , Tamanho Celular , Megacariócitos/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Contagem de Plaquetas , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Imunológicos/genética , Trombocitopenia/genética , Trombocitopenia/patologia
14.
Arterioscler Thromb Vasc Biol ; 32(12): 2956-65, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23065825

RESUMO

OBJECTIVE: The receptor-like protein-tyrosine phosphatase (PTP) CD148 and the nontransmembrane PTP1-B have been shown to be net positive regulators of Src family kinases in platelets. In the present study, we compared the relative contributions of these PTPs in platelet activation by the major glycoprotein, glycoprotein VI, α(IIb)ß(3), and C-type lectin-like receptor 2 (CLEC-2). METHODS AND RESULTS: PTP-1B-deficient mouse platelets responded normally to the glycoprotein VI-specific agonist collagen-related peptide and antibody-mediated CLEC-2 activation. However, they exhibited a marginal reduction in α(IIb)ß(3)-mediated Src family kinase activation and tyrosine phosphorylation. In contrast, CD148-deficient platelets exhibited a dramatic reduction in activation by glycoprotein VI and α(IIb)ß(3) and a marginal reduction in response to activation by CLEC-2, which was further enhanced in the absence of PTP-1B. These defects were associated with reduced activation of Src family kinase and spleen tyrosine kinase, suggesting a causal relationship. Under arteriolar flow conditions, there was defective aggregate formation in the absence of PTP-1B and, to a greater extent, CD148 and a severe abrogation of both adhesion and aggregation in the absence of both PTPs. CONCLUSIONS: Findings from this study demonstrate that CD148 plays a dominant role in activating Src family kinases in platelets relative to PTP-1B. Both PTPs are required for optimal platelet activation and aggregate formation under high arterial shear rates.


Assuntos
Plaquetas/fisiologia , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Animais , Plaquetas/citologia , Adesão Celular/fisiologia , Fibrinogênio , Glicoproteínas/fisiologia , Técnicas In Vitro , Lectinas Tipo C/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Transdução de Sinais/fisiologia , Quinases da Família src/fisiologia
15.
Microbiology (Reading) ; 144 ( Pt 8): 2263-2269, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9720049

RESUMO

Oxidation of ferrous iron was studied with the anaerobic phototrophic bacterial strain BS-1. Based on morphology, substrate utilization patterns, arrangement of intracytoplasmic membranes and the in vivo absorption spectrum, this strain was assigned to the known species Rhodomicrobium vannielii. Also, the type strain of this species oxidized ferrous iron in the light. Phototrophic growth of strain BS-1 with ferrous iron as electron donor was stimulated by the presence of acetate or succinate as cosubstrates. The ferric iron hydroxides produced precipitated on the cell surfaces as solid crusts which impeded further iron oxidation after two to three generations. The complexing agent nitrilotriacetate stimulated iron oxidation but the yield of cell mass did not increase stoichiometrically under these conditions. Other complexing agents inhibited cell growth. Ferric iron was not reduced in the dark, and manganese salts were neither oxidized nor reduced. It is concluded that ferrous iron oxidation by strain BS-1 is only a side activity of this bacterium that cannot support growth exclusively with this electron source over prolonged periods of time.


Assuntos
Compostos Ferrosos/metabolismo , Rhodospirillaceae/metabolismo , Ácido Edético/farmacologia , Ferro/metabolismo , Ácido Nitrilotriacético/farmacologia , Oxirredução/efeitos dos fármacos , Rhodobacter/metabolismo , Rhodospirillaceae/efeitos dos fármacos
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