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RATIONALE: Early steps in glomerular injury are poorly understood in collagen IV nephropathies. OBJECTIVES: We characterized structural, functional, and biophysical properties of glomerular capillaries and podocytes in Col4α3-/- mice and analyzed kidney cortex transcriptional profiles at various disease stages. We investigated the effects of TUDCA (suppresses ER stress) on these parameters and used human FSGS transcriptomic data to identify pathways rescued by TUDCA. FINDINGS: In Col4α3-/- mice, podocyte injury develops by 3 months, with maximum glomerular deformability and 40% podocyte loss at 4 months. This period is followed is followed by glomerular capillary stiffening, proteinuria, reduced renal function, inflammatory infiltrates, and fibrosis. Bulk RNA sequencing at sequential time points revealed progressive increases in inflammatory and injury gene expression, and activation of the TNF pathway. Mapping Podocyte-enriched genes from FSGS patients to mice showed that TUDCA, which mitigated renal injury suppressed molecular pathways associated with podocyte stress, hypertrophy and tubulo-interstitial injury. CONCLUSIONS: Col4α3-/- nephropathy progresses in two phases. The first is characterized by podocytopathy, increased glomerular capillary deformability and accelerated podocyte loss, and the second by increased capillary wall stiffening and renal inflammatory and profibrotic pathway activation. The response of podocytes to TUDCA treatment provides insights into signaling pathways in Alport and related nephropathies.
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Lipid metabolic reprogramming of tumor cells has been proven to play a critical role in tumor initiation and development. However, lipid metabolism in cancer-associated fibroblasts (CAFs) has rarely been studied, particularly in CAFs of oral squamous cell carcinoma (OSCC). Additionally, the molecular mechanism by which tumor cells regulate lipid metabolism in fibroblasts is unclear. In this study, we found that phosphorylated ATP citrate lyase (p-ACLY), a key lipid metabolic enzyme, was upregulated in OSCC CAFs. Compared to paracancerous normal fibroblasts, CAFs showed enhanced lipid synthesis, such as elevated cytosolic acetyl-CoA level and accumulation of lipid droplets. Conversely, reduction of p-ACLY level blocked this biological process. In addition, blocking lipid synthesis in CAFs or inhibiting fatty acid uptake by OSCC cells reduced the promotive effects of CAFs on OSCC cell proliferation, invasion, and migration. These findings suggested that CAFs are one of lipid sources required for OSCC progression. Mechanistically, AKT signaling activation was involved in the upregulation of p-ACLY level and lipid synthesis in CAFs. Interleukin-8 (IL8), an exocrine cytokine of OSCC cells, could activate AKT and then phosphorylate ACLY in fibroblasts. This study suggested that the IL8/AKT/p-ACLY axis could be considered as a potential target for OSCC treatment.
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ATP Citrato (pro-S)-Liasa , Fibroblastos Asociados al Cáncer , Carcinoma de Células Escamosas , Progresión de la Enfermedad , Interleucina-8 , Proteínas Proto-Oncogénicas c-akt , Animales , Humanos , Masculino , Ratones , ATP Citrato (pro-S)-Liasa/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Interleucina-8/metabolismo , Metabolismo de los Lípidos , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Regulación hacia ArribaRESUMEN
Despite obvious tumor shrinkage, relapse after chemotherapy remains a main cause of cancer-related mortality, indicating that a subpopulation of cancer cells acquires chemoresistance and lingers after treatment. However, the mechanism involved in the emergence of chemoresistant cells remains largely unknown. Here, we demonstrate that the degradation of mitochondria via autophagy leads to a dormant state in a subpopulation of cancer cells and confers on them resistance to lethal cisplatin (DDP) exposure. The surviving DDP-resistant cells (hereafter, DRCs) have a lower metabolic rate but a stronger potential malignant potential. In the absence of DDP, these DRCs exhibit an ever-increasing self-renewal ability and heightened tumorigenicity. The combination of chloroquine and DDP exerts potent tumor-suppressive effects. In summary, our findings illuminate the mechanism between mitophagy and tumor dormancy and prove that targeting mitophagy might be a promising approach for overcoming chemoresistance in head and neck squamous cell carcinoma (HNSCC).
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The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1ß into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.
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Given the key roles of cancer associated fibroblasts (CAFs) in shaping tumor stroma, this study shows a CAF-associated ITGB1-inactivating peptide-enriched membrane nanodelivery system (designated as PMNPs-D) to simultaneously target CAFs and tumor cells for boosted chemotherapy through promoted drug perfusion. In the structure of PMNPs-D, the PLGA-based inner core is loaded with the chemotherapeutic drug doxorubicin, and the outer surface is cloaked by hybrid biomembranes with the insertion of integrin ß1 (ITGB1) inhibiting peptide (i.e., FNIII14). After prolonged blood circulation and actively targeting in tumor sites, PMNPs-D can respond to CAF-overexpressed fibroblast activation protein-α (FAP-α) to trigger the release of FNIII14, which will bind to ITGB1 and inhibit CAFs' biological function in producing the stromal matrix, thereby loosening the condensed stromal structure and enhancing the permeability of nanotherapeutics in tumors. As a result, this tailor-designed nanosystem shows substantial tumor inhibition and metastasis retardation in aggressive adenoid cystic carcinoma (ACC) tumor-harboring mice.
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Fibroblastos Asociados al Cáncer , Neoplasias , Animales , Ratones , Fibroblastos Asociados al Cáncer/patología , Neoplasias/patología , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Membranas , Péptidos/metabolismo , Microambiente Tumoral , Línea Celular Tumoral , Fibroblastos/metabolismoRESUMEN
Cardiac muscle myosin regulatory light chain (RLC) is constitutively phosphorylated at â¼0.4 mol phosphate/mol RLC in normal hearts, and phosphorylation is maintained by balanced activities of dedicated cardiac muscle-specific myosin light chain kinase and myosin light chain phosphatase (MLCP). Previously, the identity of the cardiac-MLCP was biochemically shown to be similar to the smooth muscle MLCP, which is a well-characterized trimeric protein comprising the regulatory subunit (MYPT1), catalytic subunit PP1cß, and accessory subunit M20. In smooth muscles in vivo, MYPT1 and PP1cß co-stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, MYPT1 and MYPT2 are both expressed, but contributions to physiological regulation of cardiac myosin dephosphorylation are unclear. We hypothesized that the main catalytic subunit for cardiac-MLCP is PP1cß, and maintenance of RLC phosphorylation in vivo is dependent on regulation by striated muscle-specific MYPT2. Here, we used PP1cß conditional knockout mice to biochemically define cardiac-MLCP proteins and developed a cardiac myofibrillar phosphatase assay to measure the direct contribution of MYPT-regulated and MYPT-independent phosphatase activities toward phosphorylated cardiac myosin. We report that (1) PP1cß is the main isoform expressed in the cardiac myocyte, (2) cardiac muscle pathogenesis in PP1cß knockout animals involve upregulation of total PP1cα in myocytes and non-muscle cells, (3) the stability of cardiac MYPT1 and MYPT2 proteins in vivo is not dependent on the PP1cß expression, and (4) phosphorylated myofibrillar cardiac myosin is dephosphorylated by both myosin-targeted and soluble MYPT-independent PP1cß activities. These results contribute to our understanding of the cardiac-MLCP in vivo.
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Miosinas Cardíacas , Fosfatasa de Miosina de Cadena Ligera , Proteína Fosfatasa 1 , Animales , Miosinas Cardíacas/metabolismo , Ratones , Ratones Noqueados , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfatos/metabolismo , Fosforilación , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismoRESUMEN
Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.
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Actinas/genética , Enfermedades de la Aorta/genética , Chaperonina con TCP-1/metabolismo , Mutación Puntual , Actinas/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Ratones , Ratones Endogámicos C57BL , Mutación MissenseRESUMEN
BACKGROUND: The kidney is the source of sKlotho and kidney-specific loss of Klotho leads to a phenotype resembling the premature multiorgan failure phenotype in Klotho-hypomorphic mice ( kl/kl mice). Klotho and the Ca-sensing receptor (CaSR) are highly expressed in the distal convoluted tubule (DCT). The physiologic mechanisms that regulate sKlotho levels are unknown. METHODS: We measured sKlotho in WT and tubule-specific CaSR -/- (TS-CaSR -/- ) mice treated with calcimimetics, alkali, or acid, and Klotho shed from minced mouse kidneys, and from HEK-293 cells expressing the CaSR and Klotho, in response to calcimimetics, calcilytics, alkalotic and acidic pH, and ADAM protease inhibitors. The CaSR, Klotho, and ADAM10 were imaged in mouse kidneys and cell expression systems using confocal microscopy. RESULTS: The CaSR, Klotho, and ADAM10 colocalize on the basolateral membrane of the DCT. Calcimimetics and HCO 3 increase serum sKlotho levels in WT but not in CaSR -/- mice, and acidic pH suppresses sKlotho levels in WT mice. In minced kidneys and cultured cells, CaSR activation with high Ca, calcimimetics, or alkali increase shed Klotho levels via ADAM10, as demonstrated using the ADAM10 inhibitor GI254023X and siRNA. In cultured cells, the CaSR, Klotho, and ADAM10 form cell surface aggregates that disperse after CaSR activation. CONCLUSIONS: We identify a novel physiologic mechanism for regulation of sKlotho levels by the renal CaSR-ADAM10-Klotho pathway. We show that CaSR activators, including alkali, increase renal CaSR-stimulated Klotho shedding and predict that this mechanism is relevant to the effects of acidosis and alkali therapy on CKD progression.
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Glucuronidasa , Receptores Sensibles al Calcio , Humanos , Ratones , Animales , Receptores Sensibles al Calcio/genética , Glucuronidasa/metabolismo , Células HEK293 , Riñón/metabolismo , Proteína ADAM10 , Concentración de Iones de HidrógenoRESUMEN
Adenoid cystic carcinoma (AdCC) of the head and neck originates from salivary glands, with high risks of recurrence and metastasis that account for the poor prognosis of patients. The purpose of this research was to identify key genes related to AdCC for further investigation of their diagnostic and prognostic significance. In our study, the AdCC sample datasets GSE36820, GSE59702 and GSE88804 from the Gene Expression Omnibus (GEO) database were used to explore the abnormal coexpression of genes in AdCC compared with their expression in normal tissue. A total of 115 DEGs were obtained by screening with GEO2R and FunRich software. According to functional annotation analysis using Enrichr, these DEGs were mainly enriched in the SOX2, AR, SMAD and MAPK signaling pathways. A protein-protein network of the DEGs was established by the Search Tool for the Retrieval of Interacting Genes (STRING) and annotated through the WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) and was shown to be enriched with proteins involved in cardiac muscle cell proliferation and extracellular matrix organization. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ITGA9, LAMB1 and BAMBI were associated with the PI3K-Akt and TGF-ß pathways. Furthermore, 36 potential target miRNAs were identified by the OncomiR and miRNA Pathway Dictionary Database (miRPathDB). In conclusion, SLC22A3, FOXP2, Cdc42EP3, COL27A1, DUSP1 and HSPB8 played critical roles according to the enrichment analysis; ITGA9, LAMB1 and BAMBI were involved in significant pathways according to the KEGG analysis; ST3Gal4 is a pivotal component of the PPI network of all the DEGs obtained; SPARC, COL4A2 and PRELP were highly related to multiple malignancies in pan-cancer research; hsa-miR-29-3p, hsa-miR-132-3p and hsa-miR-708-5p were potential regulators in AdCC. The involved pathways, biological processes and miRNAs have been shown to play significant roles in the genesis, growth, invasion and metastasis of AdCC. In this study, these identified DEGs were considered to have a potential influence on AdCC but have not been studied in this disease. The analysis results promote our understanding of the molecular mechanisms and biological processes of AdCC, which might be useful for targeted therapy or diagnosis.
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Nuclear actin has been elusive due to the lack of knowledge about molecular mechanisms. From actin-containing chromatin remodeling complexes, we discovered an arginine mono-methylation mark on an evolutionarily conserved R256 residue of actin (R256me1). Actin R256 mutations in yeast affect nuclear functions and cause diseases in human. Interestingly, we show that an antibody specific for actin R256me1 preferentially stains nuclear actin over cytoplasmic actin in yeast, mouse, and human cells. We also show that actin R256me1 is regulated by protein arginine methyl transferase-5 (PRMT5) in HEK293 cells. A genome-wide survey of actin R256me1 mark provides a landscape for nuclear actin correlated with transcription. Further, gene expression and protein interaction studies uncover extensive correlations between actin R256me1 and active transcription. The discovery of actin R256me1 mark suggests a fundamental mechanism to distinguish nuclear actin from cytoplasmic actin through post-translational modification (PTM) and potentially implicates an actin PTM mark in transcription and human diseases.
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Actinas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Factores de Transcripción/metabolismo , Animales , Humanos , Metilación , RatonesRESUMEN
Cytoskeletal structure and its regulation are essential for maintenance of the differentiated state of specific types of cells and their adaptation to physiologic and pathophysiologic conditions. Renal glomerular capillaries, composed of podocytes, endothelial cells, and the glomerular basement membrane, have distinct structural and biophysical properties and are the site of injury in many glomerular diseases. Calcineurin inhibitors, immunosuppressant drugs used for organ transplantation and auto-immune diseases, can protect podocytes and glomerular capillaries from injury by preserving podocyte cytoskeletal structure. These drugs cause complications including hypertension and hyperkalemia which are mediated by WNK (With No Lysine) kinases as well as vasculopathy with glomerulopathy. WNK kinases and their target kinases oxidative stress-responsive kinase 1 (OSR1) and SPS1-related proline/alanine-rich kinase (SPAK) have fundamental roles in angiogenesis and are activated by calcineurin inhibitors, but the actions of these agents on kidney vasculature, and glomerular capillaries are not fully understood. We investigated WNK1 expression in cultured podocytes and isolated mouse glomerular capillaries to determine if WNK1 contributes to calcineurin inhibitor-induced preservation of podocyte and glomerular structure. WNK1 and OSR1/SPAK are expressed in podocytes, and in a pattern similar to podocyte synaptopodin in glomerular capillaries. Calcineurin inhibitors increased active OSR1/SPAK in glomerular capillaries, the Young's modulus (E) of glomeruli, and the F/G actin ratio, effects all blocked by WNK inhibition. In glomeruli, WNK inhibition caused reduced and irregular synaptopodin-staining, abnormal capillary and foot process structures, and increased deformability. In cultured podocytes, FK506 activated OSR1/SPAK, increased lamellipodia, accelerated cell migration, and promoted traction force. These actions of FK506 were reduced by depletion of WNK1. Collectively, these results demonstrate the importance of WNK1 in regulation of the podocyte actin cytoskeleton, biophysical properties of glomerular capillaries, and slit diaphragm structure, all of which are essential to normal kidney function.
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Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cß (PP1cß) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cß may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the ß isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cß knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cß to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cß content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cß forms in smooth muscle.
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Músculo Liso/enzimología , Proteína Fosfatasa 1/metabolismo , Animales , Íleon/enzimología , Íleon/fisiología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Liso/fisiología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/fisiología , Fosforilación , Proteína Fosfatasa 1/genética , Vejiga Urinaria/enzimología , Vejiga Urinaria/fisiologíaRESUMEN
Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.
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Queratinocitos/metabolismo , Piel/metabolismo , Adulto , Separación Celular , Células Cultivadas , Humanos , Queratinocitos/citología , Piel/citologíaRESUMEN
Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.
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Fenómenos Biofísicos , Citoesqueleto/fisiología , Glomerulonefritis/fisiopatología , Nefrosis Lipoidea/fisiopatología , Podocitos/fisiología , Animales , Adhesión Celular , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Módulo de Elasticidad , Glomerulonefritis/genética , Glomerulonefritis/patología , VIH/genética , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Nefrosis Lipoidea/inducido químicamente , Nefrosis Lipoidea/patología , Paxillin/metabolismo , Podocitos/patología , Protaminas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2 We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2 Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.
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Actinas/metabolismo , Fibroblastos/metabolismo , Mutación Missense , Piel/metabolismo , Actinas/genética , Adulto , Disección Aórtica/genética , Aorta/metabolismo , Aneurisma de la Aorta Torácica/genética , Biopsia , Dominio Catalítico , Movimiento Celular , Proliferación Celular , Niño , Citoesqueleto/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Telomerasa/genética , Transcripción GenéticaRESUMEN
Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model - all conventionally referred to as "contraction" - involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation.
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Fibroblastos/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Fenómenos Biomecánicos , Adhesión Celular/fisiología , Agregación Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , ARN Interferente Pequeño/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF-ß1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A-dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity.
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Diferenciación Celular , Dermis/lesiones , Dermis/metabolismo , Regulación de la Expresión Génica , Miofibroblastos/metabolismo , Transactivadores/metabolismo , Cicatrización de Heridas , Androstenoles/farmacología , Animales , Dermis/patología , Ratones , Miofibroblastos/patología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND & AIMS: Upon liver injury, hepatic stellate cells (HSCs) undergo dramatic morphological and functional changes including migration and contraction. In the present study, we investigated the role of myosin II isoforms in the development of the contractile phenotype of mouse HSCs, which are considered therapeutic targets to decrease portal hypertension and fibrosis. METHODS: We characterized the expression of myosin IIA and IIB in primary mouse HSCs and addressed their function by gene knock-down using isoform-specific siRNAs. RESULTS: We found that myosin IIA and IIB are differentially expressed and localized and have clearly different functions in HSCs. Myosin IIA is mainly located in the subcortical area of quiescent HSCs and at α-SMA-containing stress fibres after activation, while myosin IIB is located in the cytoplasm and at the edge of protrusions of quiescent HSCs, at stress fibres of activated cells, and at the leading edge of lamellipodia. Knock-down of myosin IIA in HSCs influences cell size and shape, results in the disruption of stress fibres and in a decrease of focal adhesions, and inhibits contractility and intra-cellular Ca(2+) release but increases cell migration. Myosin IIB contributes to the extension of lamellipodia and cell spreading but has no direct role in stress fibres and focal adhesion formation, contraction, or intra-cellular Ca(2+) signalling. CONCLUSIONS: In mouse HSCs, myosin IIA and IIB clearly fulfil distinct roles. Our results provide an insight into the contractile machinery of HSCs, that could be important in the search for new molecules to treat portal hypertension.
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Células Estrelladas Hepáticas/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Movimiento Celular/fisiología , Retículo Endoplásmico/fisiología , Endotelina-1/farmacología , Adhesiones Focales/fisiología , Técnicas de Silenciamiento del Gen , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Proteínas Motoras Moleculares/antagonistas & inhibidores , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Seudópodos/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Fibras de Estrés/fisiología , Vinculina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts. EXPERIMENTAL APPROACH: Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments. KEY RESULTS: In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration. CONCLUSIONS AND IMPLICATIONS: By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.
Asunto(s)
Células Estrelladas Hepáticas/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Miosina Tipo II/antagonistas & inhibidores , Actinas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Estrelladas Hepáticas/fisiología , Células Estrelladas Hepáticas/ultraestructura , Ratones , Miosina Tipo II/fisiología , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/ultraestructuraRESUMEN
Reactive oxygen species (ROS) are known to be involved in redox signalling pathways that may contribute to normal cell function as well as disease progression. The tumour suppressor PTEN and the inositol 5-phosphatase SHIP2 are critical enzymes in the control of PtdIns(3,4,5)P(3) level. It has been reported that oxidants, including those produced in cells such as macrophages, can activate downstream signalling via the inactivation of PTEN. The present study evaluates the potential impact of SHIP2 on phosphoinositides in cells exposed to sodium peroxide. We used a model of SHIP2 deficient mouse embryonic fibroblasts (MEFs) stimulated by H(2)O(2): at 15 min, PtdIns(3,4,5)P(3) was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P(2) could be detected at 15 or 120 min incubation of the cells with H(2)O(2) (0.6 mM). PKB activity was also upregulated in SHIP2 -/- cells as compared to +/+ cells in response to H(2)O(2). SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role as a lipid phosphatase in the control of PtdIns(3,4,5)P(3) level in response to H(2)O(2). We conclude that SHIP2 lipid phosphatase activity plays an important role in the metabolism PtdIns(3,4,5)P(3) which is demonstrated in oxygen stressed cells.