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R-Spondin 2 Is Upregulated in Idiopathic Pulmonary Fibrosis and Affects Fibroblasts Behavior
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A2195 - R-Spondin 2 Is Upregulated in Idiopathic Pulmonary Fibrosis and Affects Fibroblasts Behavior
Author Block: A. Munguia1, Y. I. Balderas-Martinez2, L. C. Becerril1, M. Checa1, B. Ortiz1, A. Pardo3, M. Selman1; 1Instituto Nacional de Enfermedades Respiratorias, Ciudad de Mexico, Mexico, 2Research, CONACYT-Instituto Nacional de Enfermedades Respiratorias Ismael CosÃo Villegas, Ciudad de Mexico, Mexico, 3Facultad De Ciencias, Univ Nacional Autonoma De Mexico, Mexico, Mexico.
RATIONALE Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblasts population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondins family (RSPO) comprises a group of proteins essential for development. From them, RSPO2 is expressed primarily in the lungs and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical WNT 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. METHODS Lung localization of RSPO2 and Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6) was examined by immunohistochemistry. IPF and normal lung fibroblasts were stimulated with rhRSPO2, and the expression profile was analyzed by RNA sequencing. Expression of gene of interests was validated by RT-PCR, and confirmed at the protein level by Western blot and Sircol assay. Cell proliferation was examined by WST-1 assay and CyQuant and cell death by Annexin V/PI assay on flow cytometry. Lost-of-function experiments were performed silencing RSPO2 gene by transfecting shRNA that specifically target the gene. RESULTS We found that RSPO2 and its receptor LGR6 were upregulated in IPF lungs where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant hRSPO2 resulted in the deregulation of numerous genes although the transcriptional response was essentially distinct. Primarily in IPF fibroblasts, several altered pathways were revealed after RSPO2 stimulation including WNT pathway (mainly from non-canonical signaling), cell cycle and apoptosis. According with the 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 (MMP)-1. Silencing RSPO2 with short hairpin RNA induced the opposite effects. CONCLUSIONS Our findings demonstrate that RSPO2 is up-regulated in IPF where it appears to have an antifibrotic role. This study was funded by CONACYT Grant #252624
Author Block: A. Munguia1, Y. I. Balderas-Martinez2, L. C. Becerril1, M. Checa1, B. Ortiz1, A. Pardo3, M. Selman1; 1Instituto Nacional de Enfermedades Respiratorias, Ciudad de Mexico, Mexico, 2Research, CONACYT-Instituto Nacional de Enfermedades Respiratorias Ismael CosÃo Villegas, Ciudad de Mexico, Mexico, 3Facultad De Ciencias, Univ Nacional Autonoma De Mexico, Mexico, Mexico.
RATIONALE Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblasts population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondins family (RSPO) comprises a group of proteins essential for development. From them, RSPO2 is expressed primarily in the lungs and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical WNT 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. METHODS Lung localization of RSPO2 and Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6) was examined by immunohistochemistry. IPF and normal lung fibroblasts were stimulated with rhRSPO2, and the expression profile was analyzed by RNA sequencing. Expression of gene of interests was validated by RT-PCR, and confirmed at the protein level by Western blot and Sircol assay. Cell proliferation was examined by WST-1 assay and CyQuant and cell death by Annexin V/PI assay on flow cytometry. Lost-of-function experiments were performed silencing RSPO2 gene by transfecting shRNA that specifically target the gene. RESULTS We found that RSPO2 and its receptor LGR6 were upregulated in IPF lungs where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant hRSPO2 resulted in the deregulation of numerous genes although the transcriptional response was essentially distinct. Primarily in IPF fibroblasts, several altered pathways were revealed after RSPO2 stimulation including WNT pathway (mainly from non-canonical signaling), cell cycle and apoptosis. According with the 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 (MMP)-1. Silencing RSPO2 with short hairpin RNA induced the opposite effects. CONCLUSIONS Our findings demonstrate that RSPO2 is up-regulated in IPF where it appears to have an antifibrotic role. This study was funded by CONACYT Grant #252624
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