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SUMMARY: The R-spondin protein family is a group of proteins that enhance Wnt/b-catenin signaling and have pleiotropic functions in stem cell growth and development. In the literature reviews, there is no histomorphological study showing the localization and distribution of R-spondins in different hypothalamic nuclei. For this reason, the purpose of this study was to determine the localization, distribution characteristics, and densities in the hypothalamic nuclei of neurons expressing Rspo1 and Rspo3 proteins. The free-floating brain sections of the male rats who were not exposed to any treatment were stained with the indirect immunoperoxidase method using the relevant antibodies. As a result of the immunohistochemical studies, it was determined that neurons expressing the Rspo1 protein were found in large numbers in the supraoptic nucleus (SON), the suprachiasmatic nucleus (SCh), anterior paraventricular nucleus, periventricular hypothalamic nucleus (PeV), anterior hypothalamic area, magnocellular preoptic nucleus (MCPO) and the lateral hypothalamic area (LH) from the hypothalamic nuclei, while they were localized in fewer numbers in the arcuate nucleus (ARC). Rspo3 protein expression was found in neurons localized in the hypothalamic nuclei SON, paraventricular nucleus (PVN), PeV, ARC, ventromedial nucleus (VMH), LH, anterior parvicellular nucleus, and zona inserta (ZI). In addition, neurons synthesizing both peptides were found in the cortex and hippocampus regions (H). Rspo1 and 3 proteins are expressed in hypothalamic energy homeostatic areas, thus these proteins may be involved in the regulation of food intake.
La familia de proteínas R-espondina es un grupo de proteínas que mejoran la señalización de Wnt/b-catenina y tienen funciones pleiotrópicas en el crecimiento y desarrollo de las células madre. En las revisiones de la literatura no existen estudios histomorfológicos que muestren la localización y distribución de las R-espondinas en diferentes núcleos hipotalámicos. Por esta razón, el propósito de este estudio fue determinar la localización, características de distribución y densidades en los núcleos hipotalámicos de neuronas que expresan las proteínas Rspo1 y Rspo3. Secciones de cerebro flotantes de ratas macho que no fueron expuestas a ningún tratamiento se tiñeron con el método de inmunoperoxidasa indirecta utilizando los anticuerpos pertinentes. Como resultado de los estudios inmunohistoquímicos, se determinó que las neuronas que expresan la proteína Rspo1 se encontraron en gran número en el núcleo supraóptico (SON), el núcleo supraquiasmático (SCh), el núcleo paraventricular anterior, el núcleo hipotalámico periventricular (PeV), el núcleo hipotalámico anterior área, núcleo preóptico magnocelular (MCPO) y el área hipotalámica lateral (LH) de los núcleos hipotalámicos, mientras que se localizaron en menor número en el núcleo arqueado (ARC). La expresión de la proteína Rspo3 se encontró en neuronas localizadas en los núcleos hipotalámicos SON, núcleo paraventricular (PVN), PeV, ARC, núcleo ventromedial (VMH), LH, núcleo parvicelular anterior y zona inserta (ZI). Además, se encontraron neuronas que sintetizan ambos péptidos en las regiones de la corteza y el hipocampo (H). Las proteínas Rspo1 y 3 se expresan en áreas homeostáticas de energía hipotalámicas, por lo que estas proteínas pueden estar involucradas en la regulación de la ingesta de alimentos.
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Animais , Masculino , Ratos , Trombospondinas/metabolismo , Hipotálamo/metabolismo , Imuno-Histoquímica , Ratos Sprague-DawleyRESUMO
Introduction: Cell therapy and tissue engineering has recently emerged as a new option for short bowel syndrome (SBS) treatment, generating tissue engineered small intestine (TESI) from organoid units (OU) and biodegradable scaffolds. The recombinant human R-Spondin 1 (rhRSPO1) protein may be a key player in this process due to its mitogenic activity in intestinal stem cells. Objective: Aiming at optimizing the TESI formation process and advancing this technology closer to the clinic, we evaluated the effects of rhRSPO1 protein on OU culture and TESI formation. Methods: Intestinal OU were isolated from C57BL/6 mice and cultured in Matrigel in the presence or absence of recombinant human rhRSPO1. Throughout the culture, OU growth and survival rates were evaluated, and cells were harvested on day 3. OU were seeded onto biodegradable scaffolds, in the presence or absence of 5 µg of rhRSPO1 and implanted into the omentum of NOD/SCID mice in order to generate TESI. The explants were harvested after 30 days, weighed, fixed in formalin and embedded in paraffin for histological analysis and immunofluorescence for different cell markers. Results: After 3 days, rhRSPO1-treated OU attained a larger size, when compared to the control group, becoming 5.7 times larger on day 6. Increased survival was observed from the second day in culture, with a 2-fold increase in OU survival between days 3 and 6. A 4.8-fold increase of non-phosphorylated ß-catenin and increased relative expression of Lgr5 mRNA in the rhRSPO1-treated group confirms activation of the canonical Wnt pathway and suggests maintenance of the OU stem cell niche and associated stemness. After 30 days of in vivo maturation, rhRSPO1-treated TESI presented a larger mass than constructs treated with saline, developing a more mature intestinal epithelium with well-formed villi and crypts. In addition, the efficiency of OU-loaded rhRSPO1-treated scaffolds significantly increased, forming TESI in 100% of the samples (N = 8), of which 40% presented maximum degree of development, as compared to 66.6% in the control group (N = 9). Conclusion: rhRSPO1 treatment improves the culture of mouse intestinal OU, increasing its size and survival in vitro, and TESI formation in vivo, increasing its mass, degree of development and engraftment.
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BACKGROUND: The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic ß-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. RESULTS: rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. CONCLUSION: Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.
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Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Trombospondinas/genética , Animais , Asparagina/metabolismo , Linhagem Celular , Cromatografia em Gel , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Trombospondinas/química , Trombospondinas/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblast population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondin family (RSPO) comprises a group of proteins essential for development. Among them, RSPO2 is expressed primarily in the lungs, and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical Wingless/int1 pathway, a critical route in the pathogenesis of IPF. Thus, the aim of this study was to examine the expression and putative role of RSPO2 in this disease. We found that RSPO2 and its receptor leucine-rich G protein-coupled receptor 6 were upregulated in IPF lungs, where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant human RSPO2 resulted in the deregulation of numerous genes, although the transcriptional response was essentially distinct. In IPF fibroblasts, RSPO2 stimulation induced the up- or downregulation of several genes involved in the Wingless/int1 pathway (mainly from noncanonical signaling). In both normal and IPF fibroblasts, RSPO2 modifies the expression of genes implicated in several pathways, including the cell cycle and apoptosis. In accordance with gene expression, the stimulation of normal and IPF fibroblasts with RSPO2 significantly reduced cell proliferation and induced cell death. RSPO2 also inhibited collagen production and increased the expression of matrix metalloproteinase 1. Silencing RSPO2 with shRNA induced the opposite effects. Our findings demonstrate, for the first time to our knowledge, that RSPO2 is upregulated in IPF, where it appears to have an antifibrotic role.
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Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Regulação para Cima/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Inativação Gênica , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinase 1 da Matriz/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/ß-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/ß-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/ß-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling.