RESUMO
Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.
Assuntos
Esclerose Lateral Amiotrófica , Linfócitos T CD8-Positivos , Células Clonais , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Células Clonais/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Técnicas de Introdução de Genes , Camundongos , Neurônios Motores/patologia , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutação , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.
Assuntos
Adenocarcinoma de Pulmão , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Células A549 , Cisplatino/farmacologia , Peróxido de Hidrogênio/farmacologia , Replicação do DNA , Dano ao DNA , Adenocarcinoma de Pulmão/genética , DNA Primase/genética , DNA Primase/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5'-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Polimorfismo de Nucleotídeo Único , Domínio Catalítico , DNA/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Humanos , Fósforo-Oxigênio Liases/genética , DNA Polimerase iotaRESUMO
The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle-specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors ß-catenin and T cell-factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of ß-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds ß-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Via de Sinalização Wnt , Animais , Proteína Axina/metabolismo , Células COS , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Proteína MyoD/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores Notch/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/farmacologia , beta Catenina/metabolismoRESUMO
The lacrimal gland (LG) develops through branching morphogenesis and produces secretions, including tears, that lubricate and protect the ocular surface. Despite the prevalence of LG disorders such as dry eye, relatively little is known about the regulation of LG development. In this study, we show that the homeobox transcription factor Barx2 is highly expressed in conjunctival epithelium, eyelids and ocular [lacrimal, harderian (HG), and meibomian (MG)] glands and is necessary for normal ocular gland and eyelid development. Barx2(-/-) mice show defective LG morphogenesis, absence of the HG, and defects in MG and eyelid fusion. Ex vivo antisense assays confirm the requirement for Barx2 in LG bud elongation and branching. Gene expression profiles reveal decreased expression of several adhesion and matrix remodeling molecules in Barx2(-/-) LGs. In culture, Barx2 regulates expression of matrix metalloproteinases (MMPs) and epithelial cell migration through the extracellular matrix. Fibroblast growth factors are crucial regulators of LG development and we show that Barx2 is required for Fgf10-induced LG bud elongation and that both Barx2 and Fgf10 cooperate in the regulation of MMPs. Together, these data suggest a mechanism for the effects of loss of Barx2 on ocular gland development. Intriguingly, salivary glands that also express a high level of Barx2 develop normally in Barx2(-/-) mice and do not show altered levels of MMPs. Thus, the function of Barx2 is specific to ocular gland development. Based on our data, we propose a functional network involving Barx2, Fgf10 and MMPs that plays an essential role in regulating branching morphogenesis of the ocular glands.
Assuntos
Matriz Extracelular/metabolismo , Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Aparelho Lacrimal/anatomia & histologia , Aparelho Lacrimal/embriologia , Aparelho Lacrimal/crescimento & desenvolvimento , Morfogênese/fisiologia , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Olho/anatomia & histologia , Olho/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2(-/-) mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2(+/-) mice. Barx2(-/-) myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation.
Assuntos
Proteínas de Homeodomínio/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , FenótipoRESUMO
X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a rare neuromuscular disorder characterized by adult-onset proximal muscle weakness and lower motor neuron degeneration. SBMA was the first human disease found to be caused by a repeat expansion mutation, as affected patients possess an expanded tract of CAG repeats, encoding polyglutamine, in the androgen receptor (AR) gene. We previously developed a conditional BAC fxAR121 transgenic mouse model of SBMA and used it to define a primary role for skeletal muscle expression of polyglutamine-expanded AR in causing the motor neuron degeneration. Here we sought to extend our understanding of SBMA disease pathophysiology and cellular basis by detailed examination and directed experimentation with the BAC fxAR121 mice. First, we evaluated BAC fxAR121 mice for non-neurological disease phenotypes recently described in human SBMA patients, and documented prominent non-alcoholic fatty liver disease, cardiomegaly, and ventricular heart wall thinning in aged male BAC fxAR121 mice. Our discovery of significant hepatic and cardiac abnormalities in SBMA mice underscores the need to evaluate human SBMA patients for signs of liver and heart disease. To directly examine the contribution of motor neuron-expressed polyQ-AR protein to SBMA neurodegeneration, we crossed BAC fxAR121 mice with two different lines of transgenic mice expressing Cre recombinase in motor neurons, and after updating characterization of SBMA phenotypes in our current BAC fxAR121 colony, we found that excision of mutant AR from motor neurons did not rescue neuromuscular or systemic disease. These findings further validate a primary role for skeletal muscle as the driver of SBMA motor neuronopathy and indicate that therapies being developed to treat patients should be delivered peripherally.
Assuntos
Atrofia Bulboespinal Ligada ao X , Camundongos , Humanos , Masculino , Animais , Idoso , Atrofia Bulboespinal Ligada ao X/metabolismo , Atrofia Bulboespinal Ligada ao X/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Neurônios Motores/metabolismo , Camundongos Transgênicos , Fenótipo , Degeneração Neural/patologiaRESUMO
Skeletal muscle has recently arisen as a regulator of central nervous system (CNS) function and aging, secreting bioactive molecules known as myokines with metabolism-modifying functions in targeted tissues, including the CNS. Here, we report the generation of a transgenic mouse with enhanced skeletal muscle lysosomal and mitochondrial function via targeted overexpression of transcription factor E-B (TFEB). We discovered that the resulting geroprotective effects in skeletal muscle reduce neuroinflammation and the accumulation of tau-associated pathological hallmarks in a mouse model of tauopathy. Muscle-specific TFEB overexpression significantly ameliorates proteotoxicity, reduces neuroinflammation, and promotes transcriptional remodeling of the aged CNS, preserving cognition and memory in aged mice. Our results implicate the maintenance of skeletal muscle function throughout aging in direct regulation of CNS health and disease and suggest that skeletal muscle originating factors may act as therapeutic targets against age-associated neurodegenerative disorders.
Assuntos
Doenças Neurodegenerativas , Camundongos , Animais , Fatores de Transcrição , Doenças Neuroinflamatórias , Músculo Esquelético , Camundongos Transgênicos , Envelhecimento , Sistema Nervoso Central , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix BásicosRESUMO
Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1-/- mice exhibit impaired muscle regeneration after injury. Panx1-/- myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.
RESUMO
The neuromuscular junction (NMJ) is a specialized synapse that is the point of connection between motor neurons and skeletal muscle. Although developmental studies have established the importance of cell-cell communication at the NMJ for the integrity and full functionality of this synapse, the contribution of this structure as a primary driver in motor neuron disease pathogenesis remains uncertain. Here, we consider the biology of the NMJ and review emerging lines of investigation that are highlighting the importance of cell-cell interaction at the NMJ in spinal muscular atrophy (SMA), X-linked spinal and bulbar muscular atrophy (SBMA), and amyotrophic lateral sclerosis (ALS). Ongoing research may reveal NMJ targets and pathways whose therapeutic modulation will help slow the progression of motor neuron disease, offering a novel treatment paradigm for ALS, SBMA, SMA, and related disorders.
Assuntos
Esclerose Lateral Amiotrófica , Doença dos Neurônios Motores , Comunicação Celular , Humanos , Neurônios Motores , Junção NeuromuscularRESUMO
Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse's feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.
Assuntos
Comportamento Animal , Custos e Análise de Custo , Análise da Marcha/métodos , Doenças Neuromusculares/fisiopatologia , Fenótipo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , CaminhadaRESUMO
Motor neuron (MN) diseases are progressive disorders resulting from degeneration of neuromuscular junctions (NMJs), which form the connection between MNs and muscle fibers. NMJ-in-a-dish models have been developed to examine human MN-associated dysfunction with disease; however such coculture models have randomly oriented myotubes with immature synapses that contract asynchronously. Mechanically patterned (MP) extracellular matrix with alternating soft and stiff stripes improves current NMJ-in-a-dish models by inducing both mouse and human myoblast durotaxis to stripes where they aligned, differentiated, and fused into patterned myotubes. Compared to conventional culture on rigid substrates or unpatterned hydrogels, MP substrates supported increased differentiation and fusion, significantly larger acetylcholine (ACh) receptor clusters, and increased expression of MuSK and Lrp4, two cell surface receptors required for NMJ formation. Robust contractions were observed when mouse myotubes were stimulated by ACh, with twitch duration and frequency most closely resembling those for mature muscle on MP substrates. Fused myotubes, when cocultured with MNs, were able to form even larger NMJs. Thus MP matrices produce more functionally active NMJs-in-a-dish, which could be used to elucidate disease pathology and facilitate drug discovery.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Junção Neuromuscular/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Proteínas Relacionadas a Receptor de LDL , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Mioblastos/citologia , Receptores Proteína Tirosina Quinases , Receptores Colinérgicos , Células-Tronco/citologiaRESUMO
In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP -1-/- mice, which represent a novel mouse model for ADDE. TSP-1-/- mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM+ ) populations sorted from mouse LGs, the c-kit+ dim/EpCAM+ /Sca1 - /CD34 - /CD45 - cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1-/- LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88-98.
Assuntos
Aparelho Lacrimal/fisiologia , Regeneração , Células-Tronco/citologia , Animais , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/citologia , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transplante de Células-Tronco , Fatores de Transcrição/metabolismoRESUMO
Purpose: Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods: The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results: Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions: Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.
Assuntos
Conexinas/genética , Células Epiteliais/transplante , Regulação da Expressão Gênica , Aparelho Lacrimal/metabolismo , Proteínas do Tecido Nervoso/genética , Síndrome de Sjogren/genética , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Conexinas/biossíntese , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/terapia , Células-Tronco/metabolismoRESUMO
AIM: To identify changes in the expression of genes coding for extracellular matrix (ECM) proteins in patients with non-inflammatory corneal disorder keratoconus (KC), patients with corneal scarring, and normal controls. MATERIALS AND METHODS: Total RNA extracted from corneal tissue of 13 KC patients, 2 patients with corneal scaring and 4 normal controls was analyzed using Human Extracellular Matrix & Adhesion Molecules Profiler PCR Array. Statistically significant changes in gene expression were identified using the Data Analysis software. RESULTS: Comparison of KC and control corneas with thresholds of 1.5 or greater fold change and a p-value of 0.05 or lower, revealed 21 differentially expressed genes, 16 genes were downregulated and 5 were upregulated. Among transcripts downregulated in KC patients we identified THBS1, ADAMTS1, SPP1, several collagens and integrins. We found TGFBI (BIGH3) gene was the most significantly upregulated transcript. CONCLUSION: Development of keratoconus results in deregulation of gene expression of extracellular matrix and adhesion molecules. CLINICAL SIGNIFICANCE: Downregulation of collagens and upregulation of TGFBI repeatedly identified in KC patients may be used as clinical markers of the disease.
RESUMO
Muscle stem cells (MuSCs) exhibit distinct behavior during successive phases of developmental myogenesis. However, how their transition to adulthood is regulated is poorly understood. Here, we show that fetal MuSCs resist progenitor specification and exhibit altered division dynamics, intrinsic features that are progressively lost postnatally. After transplantation, fetal MuSCs expand more efficiently and contribute to muscle repair. Conversely, niche colonization efficiency increases in adulthood, indicating a balance between muscle growth and stem cell pool repopulation. Gene expression profiling identified several extracellular matrix (ECM) molecules preferentially expressed in fetal MuSCs, including tenascin-C, fibronectin, and collagen VI. Loss-of-function experiments confirmed their essential and stage-specific role in regulating MuSC function. Finally, fetal-derived paracrine factors were able to enhance adult MuSC regenerative potential. Together, these findings demonstrate that MuSCs change the way in which they remodel their microenvironment to direct stem cell behavior and support the unique demands of muscle development or repair.
Assuntos
Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Animais , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Embrião de Mamíferos , Feto , Fibronectinas/genética , Fibronectinas/metabolismo , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/transplante , Transdução de Sinais , Transplante de Células-Tronco , Tenascina/genética , Tenascina/metabolismo , Cicatrização/fisiologiaRESUMO
Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal muscle repair and muscle stem cell pool repopulation.
RESUMO
PURPOSE: Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration. METHODS: Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration. RESULTS: We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation. CONCLUSIONS: Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.