RESUMO
Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA1 receptor and, to a lower extent, by also the LPA6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs.
Assuntos
Adipogenia , Fator de Crescimento do Tecido Conjuntivo , Lisofosfolipídeos , Animais , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Camundongos , Lisofosfolipídeos/metabolismo , Comunicação Celular , Transdução de Sinais , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Regulação da Expressão Gênica , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Diferenciação Celular , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Humanos , Citoesqueleto de Actina/metabolismoRESUMO
Lysophosphatidic acid (LPA) is a lysophospholipid that signals through six G-protein coupled receptors (LPARs), LPA1 to LPA6. LPA has been described as a potent modulator of fibrosis in different pathologies. In skeletal muscle, LPA increases fibrosis-related proteins and the number of fibro/adipogenic progenitors (FAPs). FAPs are the primary source of ECM-secreting myofibroblasts in acute and chronic damage. However, the effect of LPA on FAPs activation in vitro has not been explored. This study aimed to investigate FAPs' response to LPA and the downstream signaling mediators involved. Here, we demonstrated that LPA mediates FAPs activation by increasing their proliferation, expression of myofibroblasts markers, and upregulation of fibrosis-related proteins. Pretreatment with the LPA1/LPA3 antagonist Ki16425 or genetic deletion of LPA1 attenuated the LPA-induced FAPs activation, resulting in decreased expression of cyclin e1, α-SMA, and fibronectin. We also evaluated the activation of the focal adhesion kinase (FAK) in response to LPA. Our results showed that LPA induces FAK phosphorylation in FAPs. Treatment with the P-FAK inhibitor PF-228 partially prevented the induction of cell responses involved in FAPs activation, suggesting that this pathway mediates LPA signaling. FAK activation controls downstream cell signaling within the cytoplasm, such as the Hippo pathway. LPA induced the dephosphorylation of the transcriptional coactivator YAP (Yes-associated protein) and promoted direct expression of target pathway genes such as Ctgf/Ccn2 and Ccn1. The blockage of YAP transcriptional activity with Super-TDU further confirmed the role of YAP in LPA-induced FAPs activation. Finally, we demonstrated that FAK is required for LPA-dependent YAP dephosphorylation and the induction of Hippo pathway target genes. In conclusion, LPA signals through LPA1 to regulate FAPs activation by activating FAK to control the Hippo pathway.
Assuntos
Via de Sinalização Hippo , Lisofosfolipídeos , Humanos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismo , Músculo Esquelético/metabolismo , FibroseRESUMO
Several common chronic diseases, muscular dystrophies (MDs), and aging lead to progressive fibrous connective tissue (fibrosis) accumulation in skeletal muscle. Cumulative past evidence points to the role of signaling lipids such as lysophosphatidic acid (LPA) and its receptors (LPARs) in different models of fibrosis. However, the potential contribution of these molecules to the fibrotic process in skeletal muscle has not been explored. Here, we show the expression of ATX/LPA/LPARs axis components in skeletal muscle, which suggests their potential relevance for the biology of this tissue. We investigated if the skeletal muscle responds to the stimulus of intramuscular (IM) LPA injections, finding an early induction of the pro-fibrotic factor connective tissue growth factor/Cellular Communication Network factor 2 (CCN2) and extracellular matrix (ECM) proteins. Also, we found that LPA induces an increase in the number of fibro/adipogenic progenitors (FAPs), which are the primary cellular source of myofibroblasts. These effects were for the most part prevented by the inhibitor Ki16425, which inhibits the LPA receptors LPA1 and LPA3, as well as in the LPA1-KO mice. We also evaluated the in vivo activation of extracellular signal-regulated kinases (ERK 1/2), AKT, c-Jun N-terminal kinase (JNK), and Yes-asocciated protein 1 (YAP) in response to LPA. Our results show that LPA induces ERK 1/2 phosphorylation in WT muscle, but not in LPA1-KO mice. Treatment with the ERK 1/2 inhibitor U0126 prevented the induction of fibronectin in response to LPA, suggesting that this pathway is involved in LPA-induced fibrosis. Altogether, these results demonstrate that ATX/LPA/LPARs constitute a pro-fibrotic axis and suggest a possible role in muscular diseases.
Assuntos
Lisofosfolipídeos , Receptores de Ácidos Lisofosfatídicos , Animais , Proteínas da Matriz Extracelular , Fibrose , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Músculo Esquelético/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
The Kallikrein Kinin System (KKS) is a vasoactive peptide system with known functions in the maintenance of tissue homeostasis, renal function and blood pressure. The main effector peptide of KKS is Bradykinin (BK). This ligand has two receptors: a constitutive B2 receptor (B2R), which has been suggested to have anti-fibrotic effects in renal and cardiac models of fibrosis; and the inducible B1 receptor (B1R), whose expression is induced by damage and inflammation. Inflammation and fibrosis are hallmarks of Duchenne muscular dystrophy (DMD), therefore we hypothesized that the KKS may play a role in this disease. To evaluate this hypothesis we used the mdx mouse a model for DMD. We blocked the endogenous activity of the KKS by treating mdx mice with B2R antagonist (HOE-140) or B1R antagonist (DesArgLeu8BK (DALBK)) for four weeks. Both antagonists increased damage, fibrosis, TGF-ß and Smad-dependent signaling, CTGF/CCN-2 levels as well as the number of CD68 positive inflammatory cells. B2R blockade also reduced isolated muscle contraction force. These results indicate that the endogenous KKS has a protective role in the dystrophic muscle. The KKS may be a new target for future therapies to reduce inflammation and fibrosis in dystrophic muscle.
RESUMO
Este trabajo tuvo como objetivo obtener y validar un antígeno buferado de Brucella abortus para la prueba de aglutinación en placa como prueba diagnóstica de base de la brucelosis bovina. Se formularon tres lotes de antígeno a partir de la multiplicación de la cepa 99 de Brucella abortus. Se realizaron los controles de calidad correspondientes (determinación de pH, volumen celular, esterilidad, capacidad buferante) y las pruebas serológicas para la evaluación del desempeño. Se emplearon 1070 muestras de suero bovino (350 positivas y 720 negativas) previamente controladas con las pruebas de diagnóstico establecidas. Se determinó la sensibilidad y especificidad diagnóstica y relativa, los valores predictivos positivos y negativos, la eficacia y la concordancia. En los tres lotes todas las características evaluadas resultaron estar dentro de los parámetros establecidos para este tipo de producto. La especificidad y sensibilidad diagnósticas fueron de 99,5 por ciento y 100 por ciento respectivamente. El valor predictivo positivo fue de 99,1 por ciento, el valor predictivo negativo fue de 100 por ciento y la eficacia de un 99,7 por ciento. El antígeno mostró una sensibilidad y especificidad relativas de un 100 por ciento y la concordancia resultó ser clasificada como muy buena. La evaluación del desempeño arrojó resultados satisfactorios, demostrando que el método de producción empleado es factible para la obtención de un producto con adecuada eficacia(AU)
The objective of this work was to obtain and validate a buffered Brucella abortus antigen for plaque agglutination test as the basic diagnostic test for bovine brucellosis. Three batches of antigen were formulated from the multiplication of strain 99 of Brucella abortus. Quality controls (determination of pH, cell volume, sterility, buffering capacity) and serological tests for performance evaluation were performed. 1070 bovine serum samples (350 positive and 720 negative) previously tested with the established diagnostic tests were used. Diagnostic and relative sensitivity and specificity, positive predictive values and negative predictive values, efficacy and concordance were determined. In all lots the evaluated characteristics proved to be within the parameters established for this type of product. Diagnostic specificity and sensitivity were 99.5 percent and 100 percent, respectively. The positive predictive values was 99.1 percent, the negative predictive values was 100 percent and the efficacy was 99.7 percent. The antigen showed a relative sensitivity and specificity of 100 percent and the concordance was classified as very good. The performance evaluation showed satisfactory results, demonstrating that the production method used is feasible to obtain a product with adequate efficiency(AU)