RESUMEN
The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.
Asunto(s)
Mutación , Proteínas Proto-Oncogénicas c-kit/química , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Regulación Alostérica , Secuencia de Aminoácidos , Antineoplásicos/química , Benzamidas/química , Células Eucariotas/metabolismo , Células Eucariotas/patología , Humanos , Mesilato de Imatinib , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Piperazinas/química , Análisis de Componente Principal , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.
Asunto(s)
Equidae/fisiología , Espermatogénesis/fisiología , Espermatogonias/citología , Nicho de Células Madre/fisiología , Células Madre/citología , Animales , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Espermatogonias/metabolismo , Células Madre/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
The existence of murine peritoneal osteoclast precursors has been already described. Also, recent reports evidenced an interplay between B lymphocytes and osteoclasts development. B-1 cells comprise a B-lymphocyte subset that resides mostly in pleural and peritoneal cavities. It has been demonstrated that B-1 cells can differentiate into mononuclear phagocytes and form multinucleated giant cells. Based on these findings, we investigated the role of B-1 lymphocytes in bone resorption and osteoclastogenesis. In vivo experimental periodontitis induced in B-1 deficient Xid mice demonstrated that bone resorption is impaired in these animals. However, reconstitution of Xid mice with B-1 cells increased bone resorption to near Balb/c values. B-1 cell derived phagocytes express the receptor activator of nuclear factor-κB (RANK) and the macrophage colony-stimulating factor receptor (M-CSFR). When cultured with RANK-ligand (RANKL) and M-CSF, B-1 cells became tartrate resistant acid phosphatase (TRAP) positive multinucleated cells, a typical osteoclast phenotype. Lacunae formation was observed when cells were cultivated onto a calcium phosphate analog, indicating functional differentiation of B1 cells into osteoclast-like cells. The dynamics of their IgM expression showed that this lymphoid marker was downregulated along the differentiation of B-1 lymphocytes into osteoclasts. Our results unveiled the first evidence that B-1 cells have a role in osteoclastogenesis and bone resorption and offer new insights in the relationship between bone and lymphoid cells.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Transdiferenciación Celular , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Resorción Ósea , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Inmunofenotipificación , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Endogámicos BALB C , Fagocitos/citología , Fagocitos/metabolismo , Ligando RANK/farmacología , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismoRESUMEN
Trypanosoma cruzi, the aetiological agent of Chagas' disease, is able to parasitise almost any type of cell in the mammalian host. Only the macrophage, however, is potentially microbicidal for the parasite. The effectiveness of macrophages in killing T. cruzi appears to be related to the degree of activation of the cell. The macrophage colony-stimulating factor (MCSF) is a glycoprotein that promotes proliferation, differentiation and activation of macrophages. It acts through a cell-surface receptor that encodes a tyrosine kinase in its cytoplasmic domain. The MCSF receptor (MCSFR) autophosphorylates upon induction with the ligand. A three to seven-fold lower degree of phosphorylation of ligand-stimulated MCSFR in monocytes/macrophages of patients with chronic Chagas' disease compared with those of healthy donors has been previously demonstrated. Only 5-10% of the population infected with T. cruzi develop chronic Chagas' disease. Furthermore, members of this group have a functional defect of the MCSFR. I therefore decided to investigate the state of the MCSFR in patients congenitally infected with T. cruzi in whom the acute phase of the disease had subsided and who had subsequently become seronegative for Chagas' disease after treatment with Nifurtimox. I found a heterogeneous pattern of ligand-stimulated phosphorylation of MCSFR ranging from a nil to a six-fold difference within the population. Healthy children born to mothers with Chagas' disease showed a fully phosphorylated receptor protein after stimulation with MCSFR. The state of the MCSFR may correlate with the potential to develop Chagas' disease.