RESUMO
Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased G(s) signaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of G(s) signaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.
Assuntos
Osso e Ossos/metabolismo , Displasia Fibrosa Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , Aplasia Pura de Série Vermelha/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Densidade Óssea , Medula Óssea/metabolismo , Medula Óssea/patologia , Osso e Ossos/patologia , Comunicação Celular , Contagem de Células , Eritropoese/genética , Feminino , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/patologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/patologia , Osteogênese/genética , Regiões Promotoras Genéticas , Receptores 5-HT4 de Serotonina/genética , Receptores 5-HT4 de Serotonina/metabolismo , Aplasia Pura de Série Vermelha/genética , Aplasia Pura de Série Vermelha/patologia , Nicho de Células-Tronco/genéticaRESUMO
BACKGROUND: A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during ß-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction. CONCLUSIONS: These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or ß-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of ß-adrenergic-induced cardiac hypertrophy.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Cardiomegalia/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/genética , Coração/fisiopatologia , Isoproterenol/efeitos adversos , Contração Miocárdica/efeitos dos fármacos , Volume Sistólico/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Cruzamento , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Eletrocardiografia , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Coração/efeitos dos fármacos , Coração/embriologia , Masculino , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Tamanho do Órgão , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
INTRODUCTION: The controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones. METHODS: We developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1α (EF1α) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site. RESULTS: We show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria. CONCLUSIONS: Our results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1α promoter is useful for driving expression in pluripotent cells, a single copy of the EF1α promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models.