RESUMO
Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4ß1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are ß1-integrin-binding partners that regulate ß1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4ß1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4ß1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4ß1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4ß1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4ß1 activity.
Assuntos
Medula Óssea/patologia , Adesão Celular , Endotélio Vascular/patologia , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mieloma Múltiplo/patologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Western Blotting , Medula Óssea/metabolismo , Movimento Celular , Proliferação de Células , Citoplasma/metabolismo , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Citometria de Fluxo , Humanos , Integrina alfa4beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia Intravital , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microvasos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Selectina-P/genética , Selectina-P/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Talina/genética , Talina/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have examined the chromatin structure around and upstream of the transcriptional start site of the human alpha2(I) collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped approximately 20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretches nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. Characterization of the human element documented functional differences with the mouse counterpart. Enhancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3' of HS3 augmented position-independent expression of the transgene. Hence, subtle differences may characterize the regulation of mammalian alpha2(I) collagen genes by evolutionarily conserved sequences.
