Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 50
Filtrar
1.
BMC Musculoskelet Disord ; 24(1): 197, 2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36927534

RESUMO

BACKGROUND: Previous studies have shown that patients with hypercholesterolemia experience elevated levels of oxidized LDL (oxLDL), a molecule which triggers inflammation and collagenase activity. In this study we discovered novel mechanistic effects of oxLDL on tendon cells and the mediators regulating matrix remodeling by analyzing the expression and activity of related proteins and enzymes. These effects may contribute to tendon damage in patients with high cholesterol. METHODS: Isolated human tendon cells (male and female donors age 28 ± 1.4 age 37 ± 5.7, respectively) were incubated in the presence or absence of oxLDL. The influence of oxLDL on the expression level of key mRNA and proteins was examined using real time quantitative PCR, ELISA and Western blots. The activities of enzymes relevant to collagen synthesis and breakdown (lysyl oxidase and matrix metalloproteinases) were quantified using fluorometry. Finally, the isolated human tendon cells in a 3D construct were exposed to combinations of oxLDL and TGF-ß to examine their interacting effects on collagen matrix remodeling. RESULTS: The one-way ANOVA of gene expression indicates that key mRNAs including TGFB, COL1A1, DCN, and LOX were significantly reduced in human tendon cells by oxLDL while MMPs were increased. The oxLDL reduced the activity of LOX at 50 µg/ml, whereas conversely MMP activities were induced at 25 µg/ml (P ≤ 0.01). COL1A1 synthesis and TGF-ß secretion were also inhibited (P ≤ 0.05). Adding recombinant TGF-ß reversed the effects of oxLDL on the expression of collagens and LOX. OxLDL also impaired collagen matrix remodeling (P ≤ 0.01), and adding TGF-ß restored the native phenotype. CONCLUSION: Exposure to oxLDL in patients with hypercholesterolemia may adversely affect the mechanical and structural properties of tendon tissue through a direct action of oxLDL on tendon cells, including impairment of TGF-ß expression. This impairment leads to disturbed matrix remodeling and synthesis, thereby potentially leading to increased risk of acute or chronic tendon injury. Our discovery may provide an opportunity for developing effective treatments for tendon injury in hypercholesterolemia patients by targeting the TGF-ß pathway.


Assuntos
Hipercolesterolemia , Traumatismos dos Tendões , Humanos , Masculino , Feminino , Adulto , Fator de Crescimento Transformador beta/metabolismo , Colágeno/metabolismo , Tendões/metabolismo , RNA Mensageiro/metabolismo
2.
Exp Cell Res ; 361(2): 277-283, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29080796

RESUMO

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.


Assuntos
Ceramidas/farmacologia , Macrófagos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Anticorpos Neutralizantes/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ceramidas/antagonistas & inibidores , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Biochem Biophys Res Commun ; 482(1): 154-158, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27833016

RESUMO

p53 is a tumor suppressor protein which is either lost or inactivated in a large majority of tumors. The small molecule 2-phenylethynesulfonamide (PES) was originally identified as the inhibitor of p53 effects on the mitochondrial death pathway. In this report we demonstrate that p53 protein from PES-treated cells was detected in reduced mobility bands between molecular weights 95-220 kDa. Resolution of p53 aggregates on urea gel was unable to reduce the high molecular weight p53 aggregates, which were shown to be primarily located in the nucleus. Therefore, our data suggest that PES exerts its effects through covalent cross-linking and nuclear retention of p53.


Assuntos
Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/fisiologia , Sítios de Ligação/efeitos dos fármacos , Núcleo Celular/química , Reagentes de Ligações Cruzadas/administração & dosagem , Reagentes de Ligações Cruzadas/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Mitocôndrias/química , Peso Molecular , Ligação Proteica/efeitos dos fármacos , Sulfonamidas/química , Proteína Supressora de Tumor p53/química
4.
J Physiol ; 594(11): 2971-83, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26670924

RESUMO

KEY POINTS: Angiopoietin-like 4 (ANGPTL4) modulates tendon neovascularization. Cyclic loading stimulates the activity of transforming growth factor-ß and hypoxia-inducible factor 1α and thereby increases the expression and release of ANGPTL4 from human tendon cells. Targeting ANGPTL4 and its regulatory pathways is a potential avenue for regulating tendon vascularization to improve tendon healing or adaptation. ABSTRACT: The mechanisms that regulate angiogenic activity in injured or mechanically loaded tendons are poorly understood. The present study examined the potential role of angiopoietin-like 4 (ANGPTL4) in the angiogenic response of tendons subjected to repetitive mechanical loading or injury. Cyclic stretching of human tendon fibroblasts stimulated the expression and release of ANGPTL4 protein via transforming growth factor-ß (TGF-ß) and hypoxia-inducible factor 1α (HIF-1α) signalling, and the released ANGPTL4 was pro-angiogenic. Angiogenic activity was increased following ANGPTL4 injection into mouse patellar tendons, whereas the patellar tendons of ANGPTL4 knockout mice displayed reduced angiogenesis following injury. In human rotator cuff tendons, the expression of ANGPTL4 was correlated with the density of tendon endothelial cells. To our knowledge, this is the first study characterizing a role of ANGPTL4 in the tendon. ANGPTL4 may assist in the regulation of vascularity in the injured or mechanically loaded tendon. TGF-ß and HIF-1α comprise two signalling pathways that modulate the expression of ANGPTL4 by mechanically stimulated tendon fibroblasts and, in the future, these could be manipulated to influence tendon healing or adaptation.


Assuntos
Angiopoietinas/biossíntese , Fibroblastos/metabolismo , Neovascularização Fisiológica/fisiologia , Tendões/metabolismo , Suporte de Carga/fisiologia , Aminoácidos Dicarboxílicos/farmacologia , Proteína 4 Semelhante a Angiopoietina , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Tendões/efeitos dos fármacos
5.
Biochim Biophys Acta ; 1851(11): 1482-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26253821

RESUMO

The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.


Assuntos
Ceramidas/biossíntese , Macrófagos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Lipoproteínas LDL/farmacologia , Fator Estimulador de Colônias de Macrófagos/deficiência , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais , Esfingomielina Fosfodiesterase/deficiência
6.
J Cell Physiol ; 231(6): 1350-63, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26529564

RESUMO

It has long been realized that hematopoietic cells may have the capacity to trans-differentiate into non-lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans-differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast-like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type-1 collagen, and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum-free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut-offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony-stimulating factor (M-CSF). The mechanisms of M-CSF-inducing trans-differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA-1 and -3, OCT3/4, NANOG, and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M-CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications.


Assuntos
Tecido Adiposo/metabolismo , Transdiferenciação Celular , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Comunicação Parácrina , Baço/metabolismo , Adipócitos/metabolismo , Adipogenia , Tecido Adiposo/citologia , Animais , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Fenótipo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Baço/citologia
7.
Cancer Cell Int ; 15: 79, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26251638

RESUMO

BACKGROUND: Etoposide has been used clinically in cancer treatment, as well as in numerous research studies, for many years. However, there is incomplete information about its exact mechanism of action in induction of cell death. METHODS: Etoposide was compared at various concentrations to characterize the mechanisms by which it induces cell death. We investigated its effects on mouse embryonic fibroblasts (MEFs) and focused on both transcriptional and non-transcriptional responses of p53. RESULTS: Here we demonstrate that treatment of MEFs with higher concentrations of etoposide induce apoptosis and activate the transcription-dependent functions of p53. Interestingly, lower concentrations of etoposide also induced apoptosis, but without any evidence of p53-dependent transcription up-regulation. Treatment of MEFs with an inhibitor of p53, Pifithrin-α, blocked p53-dependent transcription but failed to rescue the cells from etoposide-induced apoptosis. Treatment with PES, which inhibits the mitochondrial arm of the p53 pathway inhibited etoposide-induced cell death at all concentrations tested. CONCLUSIONS: We have demonstrated that transcriptional functions of p53 are dispensable for etoposide-induced cell death. The more recently characterized effects of p53 at the mitochondria, likely involving its interactions with BCL-2 family members, are thus more important for etoposide's actions.

8.
Biochem J ; 442(1): 139-49, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22103330

RESUMO

Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X(L) and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser170, a unique site with unclear function. We provide further evidence that mutation of Ser170 to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca2+/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser170-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser170 site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Serina/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Fosforilação , Serina/genética , Proteína de Morte Celular Associada a bcl/genética
9.
J Cell Biochem ; 113(8): 2622-32, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22422640

RESUMO

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Colágeno Tipo I/metabolismo , Exonucleases/metabolismo , Fibroblastos/metabolismo , Osteonectina/metabolismo , Pele/citologia , Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Células Cultivadas , Colágeno Tipo I/genética , Exonucleases/genética , Exorribonucleases , Humanos , Imunoprecipitação , Recém-Nascido , Queratinócitos/metabolismo , Osteonectina/genética , Ligação Proteica
10.
Biochem Cell Biol ; 89(4): 387-95, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21819344

RESUMO

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.


Assuntos
Sobrevivência Celular , Lipoproteínas LDL/farmacologia , Macrófagos/fisiologia , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Apoptose , Antígenos CD36/genética , Células Cultivadas , Receptores de Lipopolissacarídeos/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo , Receptores Depuradores Classe A/genética
11.
J Lipid Res ; 51(5): 991-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19965613

RESUMO

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.


Assuntos
Cálcio/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Humanos , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
12.
Biochem Cell Biol ; 88(5): 809-18, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20921992

RESUMO

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Medula Óssea/metabolismo , Citocinas/deficiência , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Células Cultivadas , Imunoprecipitação , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Mutagênese Sítio-Dirigida , Mutação/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Arterioscler Thromb Vasc Biol ; 29(1): 92-8, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18988891

RESUMO

OBJECTIVE: Macrophage survival and proliferation is believed to be a contributing factor in the development of early atherosclerotic lesions. Oxidized low density lipoprotein (oxLDL), a key mediator in the pathogenesis of this disease, has been shown to block apoptosis in macrophages deprived of growth factor. In this report, we investigate the mechanism of oxLDL-mediated macrophage survival. METHODS AND RESULTS: OxLDL, but not native LDL (nLDL), induces an immediate and oscillatory increase in intracellular calcium ([Ca(2+)](i)). We also show that the calcium/calmodulin dependent kinase, eukaryotic elongation factor-2 kinase (eEF2 kinase), is activated in response to oxLDL, an effect that can be blocked by inhibiting calcium mobilization. Furthermore, selective inhibition of eEF2 kinase reverses the prosurvival effect of oxLDL and results in cellular apoptosis. p38 MAP kinase, a negative regulator of eEF2 kinase, is activated on growth factor withdrawal, a response that can be inhibited by oxLDL. Finally, we show that oxLDL, by activating eEF2 kinase, phosphorylates and therefore inhibits eEF2, resulting in an overall decrease in protein synthesis. CONCLUSIONS: These results indicate a novel signaling pathway in which oxLDL can block macrophage apoptosis by mobilizing calcium and activating eEF2 kinase.


Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Lipoproteínas LDL/fisiologia , Macrófagos/citologia , Animais , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Cálcio/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/farmacologia , Quinase do Fator 2 de Elongação/isolamento & purificação , Feminino , Proteínas de Choque Térmico HSP90/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Peroxidase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Sci Rep ; 10(1): 12644, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724089

RESUMO

Tendons are specialized tissues composed primarily of load-responsive fibroblasts (tenocytes) embedded in a collagen-rich extracellular matrix. Habitual mechanical loading or targeted exercise causes tendon cells to increase the stiffness of the extracellular matrix; this adaptation may occur in part through collagen synthesis or remodeling. Integrins are likely to play an important role in transmitting mechanical stimuli from the extracellular matrix to tendon cells, thereby triggering cell signaling pathways which lead to adaptive regulation of mRNA translation and protein synthesis. In this study, we discovered that mechanical stimulation of integrin ß1 leads to the phosphorylation of AKT, an event which required the presence of integrin-linked kinase (ILK). Repetitive stretching of tendon cells activates the AKT and mTOR pathways, which in turn regulates mRNA translation and collagen expression. These results support a model in which integrins are an upstream component of the mechanosensory cellular apparatus, regulating fundamental tendon cell functions relevant to exercise-induced adaptation and mechanotherapy.


Assuntos
Órgãos Bioartificiais , Colágeno/metabolismo , Integrina beta1/metabolismo , Mecanotransdução Celular , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tendões/metabolismo , Adulto , Fenômenos Biomecânicos , Sobrevivência Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Integrina beta1/genética , Masculino , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Tendões/citologia
15.
J Lipid Res ; 50(8): 1676-84, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19359704

RESUMO

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.


Assuntos
Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/farmacologia , Macrófagos Peritoneais/metabolismo , Macrófagos/metabolismo , Receptores Depuradores Classe E/metabolismo , Animais , Apoptose , Transporte Biológico , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica , Lipoproteínas LDL/sangue , Macrófagos Peritoneais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Depuradores Classe E/deficiência , Receptores Depuradores Classe E/genética
16.
Cell Signal ; 20(4): 684-94, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18249092

RESUMO

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.


Assuntos
Linfócitos B/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro , Anticorpos/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Relação Dose-Resposta Imunológica , Ativação Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Inositol Polifosfato 5-Fosfatases , Cinética , Ligantes , Linfoma de Células B/enzimologia , Linfoma de Células B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro/imunologia , Serina/metabolismo , Treonina/metabolismo , Transfecção
17.
Biochem J ; 415(3): 333-44, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18842113

RESUMO

The activation of PI3K (phosphoinositide 3-kinase) family members is a universal event in response to virtually all cytokines, growth factors and hormones. As a result of formation of PtdIns with an added phosphate at the 3 position of the inositol ring, activation of the protein kinases PDK1 (phosphoinositide-dependent kinase 1) and PKB (protein kinase B)/Akt occurs. The PI3K/PKB pathway impinges upon a remarkable array of intracellular events that influence either directly or indirectly whether or not a cell will undergo apoptosis. In this review, the many ways in which PI3K/PKB can control these processes are summarized. Not all of the events described will necessarily play a role in any one cell type, but a subset of these events is probably essential for the survival of every cell.


Assuntos
Apoptose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Forkhead/metabolismo , Humanos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
18.
Cell Signal ; 19(8): 1772-83, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17521884

RESUMO

Apoptosis is an important mechanism involved in regulating the number of macrophages present at sites of inflammation. Several lines of evidence indicate that blocking macrophage apoptosis can increase atherosclerosis. We previously reported that oxidized LDL can inhibit apoptosis in cultured bone marrow-derived macrophages. We used pertussis toxin (PTX) to test whether G protein coupled receptors are activated by oxLDL. PTX is a bacterial toxin that inhibits Gi activation by ADP-ribosylating the alpha subunit of Gi, preventing the subunit from interacting with receptors. Unexpectedly, we found that PTX by itself selectively blocks macrophage apoptosis in a dose-dependent manner. PTX acts in part by inhibiting acid sphingomyelinase activity which in turn prevents generation of ceramide, which is required for macrophage apoptosis. A Gi activator peptide, mastoparan, increased ceramide levels in macrophage and induced apoptosis, but pre-treatment with PTX partially overrode mastoparan-induced apoptosis. The anti-apoptotic effect of PTX was found to require ADP-ribosylation. PTX failed to prevent A-SMase activation or apoptosis in macrophages lacking TLR4. The anti-apoptotic effect of PTX involved the same signaling pathways as those of oxidized LDL, in that both inhibited acid sphingomyelinase, and activated the phosphoinositide 3 kinase (PI3K)/protein kinase B (PKB) pathway which leads to nuclear localization of the transcription factor NFkappaB and up-regulation of Bcl-XL. These results indicate that Gi proteins, TLR4, A-SMase and the PI3K/PKB pathway are crucial components for regulation of macrophage apoptosis.


Assuntos
Macrófagos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Animais , Células da Medula Óssea/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Fêmur/citologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fatores de Tempo
19.
Wound Repair Regen ; 16(3): 379-87, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18471256

RESUMO

We have previously demonstrated that indoleamine 2, 3-dioxygenase (IDO) expressed by dermal fibroblasts generated a tryptophan deficient environment in which immune cells, but not skin cells, undergo apoptosis. However, the mechanism by which primary skin cells such as fibroblasts and keratinocytes are resistant to this culture environment is not elucidated. Here, we asked the question of whether the activity of the general control nondepressing-2 (GCN2) kinase pathway in primary immune and skin cells is differently regulated in response to IDO-induced tryptophan deficient environment. Before addressing this question, the expression of IDO in IDO-adenoviral infected fibroblasts, as a source of IDO expression, was validated. We then demonstrated a significant immunosuppressive effect of IDO expression in primary human T cells co-cultured with IDO expressing fibroblasts in the presence of allogeneic pieces of either epidermis or full thickness skin. Evaluating the mechanism by which skin cells, but not T cells, are resistant to IDO induced low tryptophan environment, we then co-cultured IDO-expressing fibroblasts with bystander human T cells, the fibroblasts, or keratinocytes for 3 days. The results showed a significant activation of apoptotic pathway as analyzed by caspase-3 induction as well as the expression of CHOP, a downstream effector of GCN2 kinase pathway in T cells, but not in skin cells.


Assuntos
Apoptose/imunologia , Células Epiteliais/imunologia , Fibroblastos/enzimologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Pele/citologia , Linfócitos T/imunologia , Adenoviridae/genética , Western Blotting , Caspase 3 , Células Cultivadas , Técnicas de Cocultura , Expressão Gênica/imunologia , Vetores Genéticos , Humanos , Imunidade Inata/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Técnicas de Cultura de Tecidos
20.
Sports Med ; 38(2): 139-60, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18201116

RESUMO

Bone has a remarkable ability to adjust its mass and architecture in response to a wide range of loads, from low-level gravitational forces to high-level impacts. A variety of types and magnitudes of mechanical stimuli have been shown to influence human bone cell metabolism in vitro, including fluid shear, tensile and compressive strain, altered gravity and vibration. Therefore, the current article aims to synthesize in vitro data regarding the cellular mechanisms underlying the response of human bone cells to mechanical loading. Current data demonstrate commonalities in response to different types of mechanical stimuli on the one hand, along with differential activation of intracellular signalling on the other. A major unanswered question is, how do bone cells sense and distinguish between different types of load? The studies included in the present article suggest that the type and magnitude of loading may be discriminated by overlapping mechanosensory mechanisms including (i) ion channels; (ii) integrins; (iii) G-proteins; and (iv) the cytoskeleton. The downstream signalling pathways identified to date appear to overlap with known growth factor and hormone signals, providing a mechanism of interaction between systemic influences and the local mechanical environment. Finally, the data suggest that exercise should emphasize the amount of load rather than the number of repetitions.


Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/fisiologia , Exercício Físico/fisiologia , Mecanotransdução Celular/fisiologia , Osteoblastos/fisiologia , Fenômenos Biomecânicos , Fenômenos Fisiológicos Celulares , Citoesqueleto/fisiologia , Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Humanos , Integrinas/fisiologia , Canais Iônicos/fisiologia , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA