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1.
Glia ; 72(5): 916-937, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38372375

RESUMO

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Axônios , Células de Schwann , Animais , Camundongos , Ratos , Sobrevivência Celular , Células Cultivadas , Ligantes , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Células de Schwann/metabolismo , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Proteínas Recombinantes
2.
J Immunol ; 208(1): 85-96, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34810220

RESUMO

Nonpathogenic cellular prion protein (PrPC) demonstrates anti-inflammatory activity; however, the responsible mechanisms are incompletely defined. PrPC exists as a GPI-anchored membrane protein in diverse cells; however, PrPC may be released from cells by ADAM proteases or when packaged into extracellular vesicles (EVs). In this study, we show that a soluble derivative of PrPC (S-PrP) counteracts inflammatory responses triggered by pattern recognition receptors in macrophages, including TLR2, TLR4, TLR7, TLR9, NOD1, and NOD2. S-PrP also significantly attenuates the toxicity of LPS in mice. The response of macrophages to S-PrP is mediated by a receptor assembly that includes the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1). PrPC was identified in EVs isolated from human plasma. These EVs replicated the activity of S-PrP, inhibiting cytokine expression and IκBα phosphorylation in LPS-treated macrophages. The effects of plasma EVs on LPS-treated macrophages were blocked by PrPC-specific Ab, by antagonists of LRP1 and the NMDA-R, by deleting Lrp1 in macrophages, and by inhibiting Src family kinases. Phosphatidylinositol-specific phospholipase C dissociated the LPS-regulatory activity from EVs, rendering the EVs inactive as LPS inhibitors. The LPS-regulatory activity that was lost from phosphatidylinositol-specific phospholipase C-treated EVs was recovered in solution. Collectively, these results demonstrate that GPI-anchored PrPC is the essential EV component required for the observed immune regulatory activity of human plasma EVs. S-PrP and EV-associated PrPC regulate innate immunity by engaging the NMDA-R/LRP1 receptor system in macrophages. The scope of pattern recognition receptors antagonized by S-PrP suggests that released forms of PrPC may have broad anti-inflammatory activity.


Assuntos
Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Inflamação/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas PrPC/metabolismo , Receptores de N-Metil-D-Aspartato/imunologia , Animais , Células Cultivadas , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas PrPC/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo
3.
J Biol Chem ; 298(3): 101642, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35090893

RESUMO

Exosomes and other extracellular vesicles (EVs) participate in cell-cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α2-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α2-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrPC) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R-LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrPC. Finally, EVs harvested from rat astrocytes carried PrPC and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrPC with the NMDA-R-LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.


Assuntos
Vesículas Extracelulares , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Crescimento Neuronal , Proteínas Priônicas , Receptores de Lipoproteínas , Receptores de N-Metil-D-Aspartato , Animais , Vesículas Extracelulares/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , N-Metilaspartato , Células PC12 , Proteínas Priônicas/metabolismo , Ratos , Receptores de Lipoproteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
4.
J Cell Biochem ; 124(5): 743-752, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36947703

RESUMO

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.


Assuntos
Citocinas , Diabetes Mellitus Tipo 2 , Humanos , Citocinas/metabolismo , Proteínas de Membrana/metabolismo , Inativadores de Plasminogênio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Chaperona BiP do Retículo Endoplasmático , N-Metilaspartato/metabolismo , Macrófagos/metabolismo , Anticorpos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo
5.
J Biol Chem ; 295(41): 14178-14188, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788217

RESUMO

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema de Sinalização das MAP Quinases , Neuritos/metabolismo , Proteínas PrPC/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células de Schwann/metabolismo , Animais , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Neuritos/patologia , Células PC12 , Proteínas PrPC/genética , Ratos , Receptores de N-Metil-D-Aspartato/genética , Células de Schwann/patologia
6.
J Neurochem ; 152(4): 468-481, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31602645

RESUMO

Prion protein (PrPC ) localizes stably in lipid rafts microdomains and is able to recruit downstream signal transduction pathways by the interaction with promiscuous partners. Other proteins have the ability to occasionally be recruited to these specialized membrane areas, within multimolecular complexes. Among these, we highlight the presence of the low-density lipoprotein receptor-related protein 1 (LRP1), which was found localized transiently in lipid rafts, suggesting a different function of this receptor that through lipid raft becomes able to activate a signal transduction pathway triggered by specific ligands, including Tissue plasminogen activator (tPA). Since it has been reported that PrPC participates in the tPA-mediated plasminogen activation, in this study, we describe the role of lipid rafts in the recruitment and activation of downstream signal transduction pathways mediated by the interaction among tPA, PrPC and LRP1 in human neuroblastoma SK-N-BE2 cell line. Co-immunoprecipitation analysis reveals a consistent association between PrPC and GM1, as well as between LRP1 and GM1, indicating the existence of a glycosphingolipid-enriched multimolecular complex. In our cell model, knocking-down PrPC by siRNA impairs ERK phosphorylation induced by tPA. Moreover the alteration of the lipidic milieu of lipid rafts, perturbing the physical/functional interaction between PrPC and LRP1, inhibits this response. We show that LRP1 and PrPC , following tPA stimulation, may function as a system associated with lipid rafts, involved in receptor-mediated neuritogenic pathway. We suggest this as a multimolecular signaling complex, whose activity depends strictly on the integrity of lipid raft and is involved in the neuritogenic signaling.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Linhagem Celular Tumoral , Humanos
7.
J Cell Sci ; 131(14)2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29930084

RESUMO

The fibrinolysis proteinase tissue-type plasminogen activator (tPA, also known as PLAT) triggers cell signaling and regulates cell physiology. In PC12 cells, Schwann cells and macrophages, the N-methyl-D-aspartate receptor (NMDA-R) mediates tPA signaling. Plasminogen activator inhibitor-1 (PAI1, also known as SERPINE1) is a rapidly acting inhibitor of tPA enzyme activity. Although tPA-initiated cell signaling is not dependent on its enzyme active site, we show that tPA signaling is neutralized by PAI1. In PC12 cells, PAI1 blocked the ERK1/2 activation mediated by tPA as well as neurite outgrowth. In Schwann cells, PAI1 blocked tPA-mediated ERK1/2 activation and cell migration. In macrophages, PAI1 blocked the ability of tPA to inhibit IκBα phosphorylation and cytokine expression. The cell signaling activity of tPA-PAI1 complex was rescued when the complex was formed with PAI1R76E, which binds to LRP1 with decreased affinity, by pre-treating cells with the LRP1 antagonist receptor-associated protein and upon LRP1 gene silencing. The inhibitory role of LRP1 in tPA-PAI1 complex-initiated cell signaling was unanticipated given the reported role of LRP1 as an NMDA-R co-receptor in signaling responses elicited by free tPA or α2-macroglobulin. We conclude that PAI1 functions as an in-hibitor not only of the enzyme activity of tPA but also of tPA receptor-mediated activities.


Assuntos
Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Linhagem Celular , Movimento Celular , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Macrófagos/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Neurônios/metabolismo , Células PC12 , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Células de Schwann/citologia , Células de Schwann/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/genética
8.
J Neuroinflammation ; 16(1): 257, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31810478

RESUMO

BACKGROUND: Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. METHODS: Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. RESULTS: Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. CONCLUSIONS: Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.


Assuntos
Astrócitos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocinas/biossíntese , Fibrinólise/fisiologia , Fibrinolíticos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Células Cultivadas , Citocinas/genética , Fibrinólise/efeitos dos fármacos , Expressão Gênica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasminogênio/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Quinazolinas/farmacologia
9.
Blood ; 130(11): 1364-1374, 2017 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-28684538

RESUMO

Tissue-type plasminogen activator (tPA) is the major intravascular activator of fibrinolysis and a ligand for receptors involved in cell signaling. In cultured macrophages, tPA inhibits the response to lipopolysaccharide (LPS) by a pathway that apparently requires low-density lipoprotein receptor-related protein-1 (LRP1). Herein, we show that the mechanism by which tPA neutralizes LPS involves rapid reversal of IκBα phosphorylation. tPA independently induced transient IκBα phosphorylation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages; however, these events did not trigger inflammatory mediator expression. The tPA signaling response was distinguished from the signature of signaling events elicited by proinflammatory LRP1 ligands, such as receptor-associated protein (RAP), which included sustained IκBα phosphorylation and activation of all 3 MAP kinases (ERK1/2, c-Jun kinase, and p38 MAP kinase). Enzymatically active and inactive tPA demonstrated similar immune modulatory activity. Intravascular administration of enzymatically inactive tPA in mice blocked the toxicity of LPS. In mice not treated with exogenous tPA, the plasma concentration of endogenous tPA increased 3-fold in response to LPS, to 116 ± 15 pM, but remained below the approximate threshold for eliciting anti-inflammatory cell signaling in macrophages (∼2.0 nM). This threshold is readily achieved in patients when tPA is administered therapeutically for stroke. In addition to LRP1, we demonstrate that the N-methyl-D-aspartic acid receptor (NMDA-R) is expressed by macrophages and essential for anti-inflammatory cell signaling and regulation of cytokine expression by tPA. The NMDA-R and Toll-like receptor-4 were not required for proinflammatory RAP signaling. By mediating the tPA response in macrophages, the NMDA-R provides a pathway by which the fibrinolysis system may regulate innate immunity.


Assuntos
Imunidade Inata/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Ligantes , Lipopolissacarídeos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Proc Natl Acad Sci U S A ; 113(5): 1369-74, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26787872

RESUMO

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment.


Assuntos
Inflamação/prevenção & controle , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Macrófagos/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Animais , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos
11.
FASEB J ; 31(4): 1744-1755, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28073836

RESUMO

In the peripheral nervous system, Schwann cells (SCs) demonstrate surveillance activity, detecting injury and undergoing trans-differentiation to support repair. SC receptors that detect peripheral nervous system injury remain incompletely understood. We used RT-PCR to profile ionotropic glutamate receptor expression in cultured SCs. We identified subunits required for assembly of N-methyl-d-aspartic acid (NMDA) receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and kainate receptors. Treatment of SCs with 40-100 µM glutamate or with 0.5-1.0 µM NMDA robustly activated Akt and ERK1/2. The response was transient and bimodal; glutamate concentrations that exceeded 250 µM failed to activate cell signaling. Phosphoprotein profiling identified diverse phosphorylated proteins in glutamate-treated SCs in addition to ERK1/2 and Akt, including p70 S6-kinase, glycogen synthase kinase-3, ribosomal S6 kinase, c-Jun, and cAMP response element binding protein. Activation of SC signaling by glutamate was blocked by EGTA and dizocilpine and by silencing expression of the NMDA-R NR1 subunit. Phosphoinositide 3-kinase/PI3K functioned as an essential upstream activator of Akt and ERK1/2 in glutamate-treated SCs. When glutamate or NMDA was injected directly into crush-injured rat sciatic nerves, ERK1/2 phosphorylation was observed in myelinated and nonmyelinating SCs. Glutamate promoted SC migration by a pathway that required PI3K and ERK1/2. These results identified ionotropic glutamate receptors and NMDA-Rs, specifically, as potentially important cell signaling receptors in SCs.-Campana, W. M., Mantuano, E., Azmoon, P., Henry, K., Banki, M. A., Kim, J. H., Pizzo, D. P., Gonias, S. L. Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.


Assuntos
Ácido Glutâmico/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácido Glutâmico/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/metabolismo , Células de Schwann/efeitos dos fármacos
12.
J Cell Sci ; 128(18): 3478-88, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26272917

RESUMO

NMDA receptors (NMDA-Rs) are ionotropic glutamate receptors, which associate with LDL-receptor-related protein-1 (LRP1) to trigger cell signaling in response to protein ligands in neurons. Here, we demonstrate for the first time that the NMDA-R is expressed by rat Schwann cells and functions independently and with LRP1 to regulate Schwann cell physiology. The NR1 (encoded by GRIN1) and NR2b (encoded by GRIN2B) NMDA-R subunits were expressed by cultured Schwann cells and upregulated in sciatic nerves following crush injury. The ability of LRP1 ligands to activate ERK1/2 (also known as MAPK3 and MAPK1, respectively) and promote Schwann cell migration required the NMDA-R. NR1 gene silencing compromised Schwann cell survival. Injection of the LRP1 ligands tissue-type plasminogen activator (tPA, also known as PLAT) or MMP9-PEX into crush-injured sciatic nerves activated ERK1/2 in Schwann cells in vivo, and the response was blocked by systemic treatment with the NMDA-R inhibitor MK801. tPA was unique among the LRP1 ligands examined because tPA activated cell signaling and promoted Schwann cell migration by interacting with the NMDA-R independently of LRP1, albeit with delayed kinetics. These results define the NMDA-R as a Schwann cell signaling receptor for protein ligands and a major regulator of Schwann cell physiology, which may be particularly important in peripheral nervous system (PNS) injury.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células de Schwann/fisiologia , Animais , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Nervo Isquiático , Transdução de Sinais , Ativador de Plasminogênio Tecidual/metabolismo
13.
Mol Cell Neurosci ; 76: 42-51, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27565578

RESUMO

LDL Receptor-related Protein-1 (LRP1) is an endocytic receptor for diverse ligands. In neurons and neuron-like cells, ligand-binding to LRP1 initiates cell-signaling. Herein, we show that in PC12 and N2a neuron-like cells, LRP1 distributes into lipid rafts and non-raft plasma membrane fractions. When lipid rafts were disrupted, using methyl-ß-cyclodextrin or fumonisin B1, activation of Src family kinases and ERK1/2 by the LRP1 ligands, tissue-type plasminogen activator and activated α2-macroglobulin, was blocked. Biological consequences of activated LRP1 signaling, including neurite outgrowth and cell growth, also were blocked. The effects of lipid raft disruption on ERK1/2 activation and neurite outgrowth, in response to LRP1 ligands, were reproduced in experiments with cerebellar granule neurons in primary culture. Because the reagents used to disrupt lipid rafts may have effects on the composition of the plasma membrane outside lipid rafts, we studied the effects of these reagents on LRP1 activities unrelated to cell-signaling. Lipid raft disruption did not affect the total ligand binding capacity of LRP1, the affinity of LRP1 for its ligands, or its endocytic activity. These results demonstrate that well described activities of LRP1 require localization of this receptor to distinct plasma membrane microdomains.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Células Cultivadas , Endocitose , Fumonisinas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Ratos , Ratos Sprague-Dawley , Ativador de Plasminogênio Tecidual/metabolismo , beta-Ciclodextrinas/farmacologia , Quinases da Família src/metabolismo
14.
J Cell Sci ; 126(Pt 1): 209-20, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23132925

RESUMO

In the injured adult mammalian central nervous system (CNS), products are generated that inhibit neuronal sprouting and regeneration. In recent years, most attention has focused on the myelin-associated inhibitory proteins (MAIs) Nogo-A, OMgp, and myelin-associated glycoprotein (MAG). Binding of MAIs to neuronal cell-surface receptors leads to activation of RhoA, growth cone collapse, and neurite outgrowth inhibition. In the present study, we identify low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) as a high-affinity, endocytic receptor for MAG. In contrast with previously identified MAG receptors, binding of MAG to LRP1 occurs independently of terminal sialic acids. In primary neurons, functional inactivation of LRP1 with receptor-associated protein, depletion by RNA interference (RNAi) knock-down, or LRP1 gene deletion is sufficient to significantly reverse MAG and myelin-mediated inhibition of neurite outgrowth. Similar results are observed when LRP1 is antagonized in PC12 and N2a cells. By contrast, inhibiting LRP1 does not attenuate inhibition of neurite outgrowth caused by chondroitin sulfate proteoglycans. Mechanistic studies in N2a cells showed that LRP1 and p75NTR associate in a MAG-dependent manner and that MAG-mediated activation of RhoA may involve both LRP1 and p75NTR. LRP1 derivatives that include the complement-like repeat clusters CII and CIV bind MAG and other MAIs. When CII and CIV were expressed as Fc-fusion proteins, these proteins, purified full-length LRP1 and shed LRP1 all attenuated the inhibition of neurite outgrowth caused by MAG and CNS myelin in primary neurons. Collectively, our studies identify LRP1 as a novel MAG receptor that functions in neurite outgrowth inhibition.


Assuntos
Sistema Nervoso Central/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuritos/metabolismo , Animais , Células CHO , Células COS , Linhagem Celular , Cricetinae , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Espectrometria de Massas , Bainha de Mielina/genética , Glicoproteína Associada a Mielina/genética , Proteínas do Tecido Nervoso , Células PC12 , Ligação Proteica , Ratos , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo
15.
J Neurosci ; 33(13): 5590-602, 2013 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-23536074

RESUMO

Trophic support and myelination of axons by Schwann cells in the PNS are essential for normal nerve function. Herein, we show that deletion of the LDL receptor-related protein-1 (LRP1) gene in Schwann cells (scLRP1(-/-)) induces abnormalities in axon myelination and in ensheathment of axons by nonmyelinating Schwann cells in Remak bundles. These anatomical changes in the PNS were associated with mechanical allodynia, even in the absence of nerve injury. In response to crush injury, sciatic nerves in scLRP1(-/-) mice showed accelerated degeneration and Schwann cell death. Remyelinated axons were evident 20 d after crush injury in control mice, yet were largely absent in scLRP1(-/-) mice. In the partial nerve ligation model, scLRP1(-/-) mice demonstrated significantly increased and sustained mechanical allodynia and loss of motor function. Evidence for central sensitization in pain processing included increased p38MAPK activation and activation of microglia in the spinal cord. These studies identify LRP1 as an essential mediator of normal Schwann cell-axonal interactions and as a pivotal regulator of the Schwann cell response to PNS injury in vivo. Mice in which LRP1 is deficient in Schwann cells represent a model for studying how abnormalities in Schwann cell physiology may facilitate and sustain chronic pain.


Assuntos
Axônios/fisiologia , Axônios/ultraestrutura , Receptores de LDL/metabolismo , Células de Schwann/patologia , Ciática/patologia , Ciática/prevenção & controle , Proteínas Supressoras de Tumor/metabolismo , Actinas/metabolismo , Análise de Variância , Animais , Antígeno CD11b/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hiperalgesia/etiologia , Hiperalgesia/genética , Marcação In Situ das Extremidades Cortadas , Indóis , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Proteína Básica da Mielina/metabolismo , Degeneração Neural/etiologia , Degeneração Neural/genética , Medição da Dor , Fosforilação/genética , Células do Corno Posterior/patologia , Células do Corno Posterior/ultraestrutura , Receptores de LDL/deficiência , Proteínas S100/metabolismo , Células de Schwann/ultraestrutura , Ciática/complicações , Ciática/genética , Transtornos de Sensação/etiologia , Medula Espinal/patologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Biol Chem ; 288(47): 34009-34018, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24129569

RESUMO

In addition to functioning as an activator of fibrinolysis, tissue-type plasminogen activator (tPA) interacts with neurons and regulates multiple aspects of neuronal cell physiology. In this study, we examined the mechanism by which tPA initiates cell signaling in PC12 and N2a neuron-like cells. We demonstrate that enzymatically active and inactive tPA (EI-tPA) activate ERK1/2 in a biphasic manner. Rapid ERK1/2 activation is dependent on LDL receptor-related protein-1 (LRP1). In the second phase, ERK1/2 is activated by tPA independently of LRP1. The length of the LRP1-dependent phase varied inversely with the tPA concentration. Rapid ERK1/2 activation in response to EI-tPA and activated α2-macroglobulin (α2M*) required the NMDA receptor and Trk receptors, which assemble with LRP1 into a single pathway. Assembly of this signaling system may have been facilitated by the bifunctional adapter protein, PSD-95, which associated with LRP1 selectively in cells treated with EI-tPA or α2M*. Myelin-associated glycoprotein binds to LRP1 with high affinity but failed to induce phosphorylation of TrkA or ERK1/2. Instead, myelin-associated glycoprotein recruited p75 neurotrophin receptor (p75NTR) into a complex with LRP1 and activated RhoA. p75NTR was not recruited by other LRP1 ligands, including EI-tPA and α2M*. Lactoferrin functioned as an LRP1 signaling antagonist, inhibiting Trk receptor phosphorylation and ERK1/2 activation in response to EI-tPA. These results demonstrate that LRP1-initiated cell signaling is ligand-dependent. Proteins that activate cell signaling by binding to LRP1 assemble different co-receptor systems. Ligand-specific co-receptor recruitment provides a mechanism by which one receptor, LRP1, may trigger different signaling responses.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Glicoproteína Associada a Mielina/metabolismo , Receptores de LDL/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lactoferrina/genética , Lactoferrina/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Glicoproteína Associada a Mielina/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Ratos , Receptores de Fatores de Crescimento , Receptores de LDL/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Ativador de Plasminogênio Tecidual/genética , Proteínas Supressoras de Tumor/genética , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismo
17.
JCI Insight ; 8(15)2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-37368488

RESUMO

Low-density lipoprotein receptor-related protein-1 (LRP1) functions as a receptor for nonpathogenic cellular prion protein (PrPC), which is released from cells by ADAM (a disintegrin and metalloproteinase domain) proteases or in extracellular vesicles. This interaction activates cell signaling and attenuates inflammatory responses. We screened 14-mer PrPC-derived peptides and identified a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111. A synthetic peptide (P3) corresponding to this region replicated the cell-signaling and biological activities of full-length shed PrPC. P3 blocked LPS-elicited cytokine expression in macrophages and microglia and rescued the heightened sensitivity to LPS in mice in which the PrPC gene (Prnp) had been deleted. P3 activated ERK1/2 and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA receptor and was blocked by the PrPC-specific antibody, POM2. P3 has Lys residues, which are typically necessary for LRP1 binding. Converting Lys100 and Lys103 into Ala eliminated the activity of P3, suggesting that these residues are essential in the LRP1-binding motif. A P3 derivative in which Lys105 and Lys109 were converted into Ala retained activity. We conclude that the biological activities of shed PrPC, attributed to interaction with LRP1, are retained in synthetic peptides, which may be templates for therapeutics development.


Assuntos
Príons , Receptores de Lipoproteínas , Ratos , Camundongos , Animais , Proteínas Priônicas , Lipopolissacarídeos , Transdução de Sinais , Príons/metabolismo , Células PC12
18.
J Neurosci ; 31(38): 13376-85, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21940431

RESUMO

In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein (CHOP), a proapoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1 signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase target eIF2α was phosphorylated and CHOP was induced by (1) inhibiting PI3K, (2) treating the cells with tumor necrosis factor-α (TNF-α), or (3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on phosphorylated eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and, by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.


Assuntos
Sobrevivência Celular/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Células de Schwann/fisiologia , Nervo Isquiático/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Morte Celular/genética , Morte Celular/fisiologia , Sobrevivência Celular/genética , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Cultura Primária de Células , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genética , Fator de Necrose Tumoral alfa/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
19.
Sci Rep ; 12(1): 17594, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266319

RESUMO

LDL Receptor-related Protein-1 (LRP1/CD91) binds diverse ligands, many of which activate cell-signaling. Herein, we compared three LRP1 ligands that inhibit inflammatory responses triggered by lipopolysaccharide (LPS), including: enzymatically-inactive tissue-type plasminogen activator (EI-tPA); activated α2-macroglobulin (α2M); and S-PrP, a soluble derivative of nonpathogenic cellular prion protein (PrPC). In bone marrow-derived macrophages, the N-methyl-D-aspartate receptor was essential for all three LRP1 ligands to activate cell-signaling and inhibit LPS-induced cytokine expression. Intact lipid rafts also were essential. Only α2M absolutely required LRP1. LRP1 decreased the EI-tPA concentration required to activate cell-signaling and antagonize LPS but was not essential, mimicking its role as a S-PrP co-receptor. Membrane-anchored PrPC also functioned as a co-receptor for EI-tPA and α2M, decreasing the ligand concentration required for cell-signaling and LPS antagonism; however, when the concentration of EI-tPA or α2M was sufficiently increased, cell-signaling and LPS antagonism occurred independently of PrPC. S-PrP is the only LRP1 ligand in this group that activated cell-signaling independently of membrane-anchored PrPC. EI-tPA, α2M, and S-PrP inhibited LPS-induced LRP1 shedding from macrophages, a process that converts LRP1 into a pro-inflammatory product. Differences in the co-receptors required for anti-inflammatory activity may explain why LRP1 ligands vary in ability to target macrophages in different differentiation states.


Assuntos
Lipopolissacarídeos , alfa 2-Macroglobulinas Associadas à Gravidez , Gravidez , Feminino , Humanos , Ligantes , Proteínas Priônicas/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Citocinas/metabolismo
20.
J Biol Chem ; 285(19): 14259-66, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20197276

RESUMO

LDL receptor-related protein (LRP1) is expressed by Schwann cells in vivo mainly after injury to the peripheral nervous system (PNS). Schwann cells in primary culture, which provide a model of Schwann cells in the injured PNS, also express abundant LRP1. Herein, we show that LRP1 gene-silencing or treatment with receptor-associated protein (RAP) promotes Schwann cell adhesion and inhibits cell migration on fibronectin. LRP1 gene-silencing also resulted in the formation of prominent focal adhesions and actin stress fibers. These changes, which were induced by loss of LRP1 expression or activity, were explained mechanistically by an increase in activated RhoA, coupled with a decrease in activated Rac1. Known LRP1 ligands, including matrix metalloprotease-9, tissue-type plasminogen activator, and alpha(2)-macroglobulin activated Rac1 in LRP1-expressing Schwann cells. An inhibitor of Rac1 activation promoted Schwann cell adhesion. Conversely, in cells in which LRP1 was silenced, a Rho kinase inhibitor promoted migration and inhibited adhesion. These results demonstrate that direct binding of ligands to LRP1 controls activation of small Rho family GTPases. The effects of LRP1 gene-silencing and RAP implicate autocrine pathways involving endogenously produced LRP1 ligands. Regulation of Schwann cell migration by LRP1 may be important in PNS injury.


Assuntos
Adesão Celular , Movimento Celular , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Células de Schwann/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Imunofluorescência , Adesões Focais/metabolismo , Inativação Gênica , Immunoblotting , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Ratos , Ratos Sprague-Dawley
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