RESUMO
Tooth movement is the result of periodontal tissue reconstruction. The biomechanical effects produced by orthopedic forces can affect the cytoskeletal rearrangement of human periodontal ligament cells (hPDLCs). However, the mechanisms responsible for the cytoskeletal rearrangement are not completely understood. To analyze the effect, we investigated the role of the Rho-mDia1 signaling pathway in cyclic strain-induced cytoskeletal rearrangement of hPDLCs in detail. We cultured hPDLCs on collagen I-coated six-well Bioflex plates and then exposed them to cyclic strain with physiological loading (10%) at a frequency of 0.1Hz for 6 or 24h using a Flexercell Tension Plus system. Notably, the cells cultured on the Bioflex plates showed increased expression levels of RhoA-GTP, profilin-1 protein, and the combination of RhoA and mDia1, whereas the expression levels of Rho-GDIa were reduced compared with a static control group. Furthermore, the cytoskeletal rearrangement of cells was enhanced. However, profilin-1 protein expression and cytoskeletal reorganization under cyclic strain can decrease due to the overexpression of Rho-GDIa or mDia1-siRNA transfection, whereas Rho-GDIa siRNA transfection has the opposite effect on hPDLCs. Together, our results demonstrate that the Rho-mDia1 signaling pathway is involved in the cytoskeletal rearrangement of hPDLCs induced by cyclic strain. These observations may enable a more in-depth understanding of orthodontic tooth movement and the reconstruction of PDL and alveolar bone.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto/ultraestrutura , Proteínas rho de Ligação ao GTP/metabolismo , Fenômenos Biomecânicos , Células Cultivadas , Citoesqueleto/metabolismo , Forminas , Humanos , Ligamento Periodontal/citologia , Transdução de SinaisRESUMO
Members of the perilipin family of lipid droplet scaffold proteins are thought to play important roles in tissue-specific regulation of triglyceride metabolism, but the mechanisms involved are not fully understood. Present results indicate that adipose triglyceride lipase (Atgl) interacts with perilipin-5 (Plin5) but not perilipin-1 (Plin1). Protein interaction assays in live cells and in situ binding experiments showed that Atgl and its protein activator, α-ß-hydrolase domain-containing 5 (Abhd5), each bind Plin5. Surprisingly, competition experiments indicated that individual Plin5 molecules bind Atgl or Abhd5 but not both simultaneously. Thus, the ability of Plin5 to concentrate these proteins at droplet surfaces involves binding to different Plin5 molecules, possibly in an oligomeric complex. The association of Plin5-Abhd5 complexes on lipid droplet surfaces was more stable than Plin5-Atgl complexes, and oleic acid treatment selectively promoted the interaction of Plin5 and Abhd5. Analysis of chimeric and mutant perilipin proteins demonstrated that amino acids 200-463 are necessary and sufficient to bind both Atgl and Abhd5 and that the C-terminal 64 amino acids of Plin5 are critical for the differential binding of Atgl to Plin5 and Plin1. Mutant Plin5 that binds Abhd5 but not Atgl was defective in preventing neutral lipid accumulation compared with wild type Plin5, indicating that the ability of Plin5 to concentrate these proteins on lipid droplets is critical to functional Atgl activity in cells.
Assuntos
Lipase/metabolismo , Proteínas/metabolismo , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Animais , Células COS , Proteínas de Transporte , Chlorocebus aethiops , Humanos , Lipase/genética , Masculino , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteínas/genética , Triglicerídeos/genéticaRESUMO
Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.
Assuntos
Esterases/fisiologia , Metabolismo dos Lipídeos , Proteínas/fisiologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase , Células 3T3 , Animais , Células Cultivadas , Esterases/genética , Esterases/metabolismo , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Camundongos , Miocárdio/metabolismo , Perilipina-5 , Ligação Proteica , Proteínas/genética , Proteínas/metabolismoRESUMO
This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.
Assuntos
Adipócitos/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Esterases/metabolismo , Lipase/metabolismo , Fosfolipases A/metabolismo , Fosfoproteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase , Células 3T3 , Adipócitos/citologia , Animais , Proteínas de Transporte , AMP Cíclico/farmacologia , Estruturas Citoplasmáticas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Camundongos , Perilipina-1 , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
Chronic activation of adipocyte beta-adrenergic receptors induces remodeling of white adipose tissue (WAT) that includes a transient inflammatory response followed by mitochondrial biogenesis, induction of fatty acid oxidation genes, and elevation of tissue oxidative metabolism. Gene profiling experiments of WAT during remodeling induced by the beta(3)-adrenergic receptor agonist CL-316,243 (CL) suggested that peroxisome proliferator-activated receptor-alpha (Ppara), which is upregulated by CL, might be an important transcriptional regulator of that process. Histological, physiological, and molecular analysis of CL-induced remodeling in wild-type mice and mice lacking Ppara demonstrated that Ppara was important for inducing adipocyte mitochondrial biogenesis and upregulating genes involved in fatty acid oxidation. Furthermore, Ppara-deficient mice exhibited sustained WAT inflammation during CL treatment, indicating that upregulation of Ppara limits proinflammatory signaling during chronic lipolytic activation. Together, these data support the hypothesis that WAT remodeling is an adaptive response to excessive fatty acid mobilization whereby Ppara and its downstream targets elevate fatty acid catabolism and suppress proinflammatory signaling.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Agonistas de Receptores Adrenérgicos beta 3 , Dioxóis/administração & dosagem , Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , PPAR alfa/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Adaptação Fisiológica/fisiologia , Tecido Adiposo/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestruturaRESUMO
Selective agonists of beta(3)-adrenergic receptors (Adrb3) exhibit potent anti-diabetes properties in rodent models when given chronically, yet the mechanisms involved are poorly understood. A salient feature of chronic Adrb3 activation is pronounced remodeling of white adipose tissue (WAT), which includes mitochondrial biogenesis and elevation of metabolic rate. To gain insights into potential mechanisms underlying WAT remodeling, the time course of remodeling induced by the Adrb3 agonist CL-316,243 (CL) was analyzed using histological, physiological, and global gene profiling approaches. The results indicate that continuous CL treatment induced a transient proinflammatory response that was followed by cellular proliferation among stromal cells and multilocular adipocytes. CL treatment strongly fragmented the central lipid storage droplet of mature adipocytes and induced mitochondrial biogenesis within these cells. Mitochondrial biogenesis was correlated with the upregulation of genes involved in fatty acid oxidation and mitochondrial electron transport activity. The elevated catabolic activity of WAT was temporally correlated with upregulation of peroxisome proliferator-activated receptor-alpha and its target genes, suggesting involvement of this transcription factor in coordinating the gene program that elevates WAT catabolic activity.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Agonistas de Receptores Adrenérgicos beta 3 , Dioxóis/administração & dosagem , Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , Receptores Adrenérgicos beta 3/metabolismo , Adaptação Fisiológica/fisiologia , Tecido Adiposo/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestruturaRESUMO
Diabetes with hypertension is characterized by increased cerebrovascular pathology and poorer outcomes following stroke. In this study we evaluated the effect of global brain ischemia on brain metabolic parameters in normal and diabetic rats treated with a dihydropyridine calcium antagonist, felodipine. Normal and diabetic rats were treated daily with felodipine (5 mg/kg) or saline. After 4 wk global ischemia was produced by occluding the carotid arteries for 1 h. In other groups the occlusion was removed and the animals were allowed to reperfuse for an additional 2 h. Following 1 h global ischemia, with or without reperfusion, the animals and controls were killed by decapitation. Cerebral water, lactate, ATP, and glutamate were measured. Glo-bal ischemia with or without reperfusion increased cerebral water and lactate, but decreased ATP. Treatment with felodipine decreased lactate, but increased water content. Ischemia in diabetics with or without reperfusion decreased water and lactate. Treated diabetics had higher ATP levels after reperfusion. Glutamate levels were increased in diabetics and were further increased by treatment. We conclude that the enhanced CNS damage following cerebral ischemia in diabetes is not correlated with ATP or lactate levels and may be mediated in part by increased glutamate. Calcium channel antagonist may augment this process.