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OBJECTIVE: The Cancer Genome Atlas (TCGA) project identified favorable prognosis regarding the ultra-mutated endometrial cancer (EC) subtype linked to polymerase epsilon gene (POLE) mutations. This study investigated POLE mutations in EC of Indian patients. METHODS: This retrospective analytical study was conducted between January 2016 and January 2023 at the Government Medical College, Kozhikode, and the MVR Cancer Center, Kozhikode, Kerala. Sanger sequencing of POLE gene exons 9 and 13 in 151 EC patients was carried out to analyze the relationship between mutations and epidemiological factors, clinicopathologic features, and treatment outcomes. RESULTS: Among 151 cases enrolled, 39 were unique POLE-mutated cases. Significant associations were high-grade tumors, myometrial invasion >50%, and Lymph-vascular space invasion (LVSI). The median follow-up was 40 months (95% confidence interval [CI], 34-46). A lower mean disease-specific survival (DSS) of 51.7 months (95% CI, 43.7-59.6) was noted in the POLE-mutated group compared with 72.11 months (95% CI, 67.60-76.62) for the POLE wild-type. A statistically significant hazard ratio (HR) of 2.683 for DSS in the POLE-mutated group was noted. In advanced stages (FIGO stages II-IV), a nine-fold HR for DSS and overall survival (OS) compared with POLE wild-type was identified. After controlling for treatment effects using Cox proportional HR, advanced-stage POLE-mutated tumors had a significantly higher HR of 8.67 for DSS compared with POLE-wild-type tumors of the same stage. CONCLUSION: This study identified a unique set of POLE mutations in Indian EC patients associated with poor prognosis, which were particularly pronounced in advanced stages. Advanced stage of presentation, type of POLE mutations, and possibly ethnicity are predictors of adverse outcomes in POLE-mutated EC. The present study highlights ethnicity as a determinant of phenotypic expression of genetic change.
Assuntos
DNA Polimerase II , Neoplasias do Endométrio , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Humanos , Feminino , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/mortalidade , Índia , DNA Polimerase II/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Prognóstico , Proteínas de Ligação a Poli-ADP-Ribose/genética , Idoso , Adulto , Prevalência , Estadiamento de NeoplasiasRESUMO
BACKGROUND: Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. OBJECTIVE: This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. MATERIALS AND METHODS: We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and in situ imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aß42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alveolar bone loss and in situ osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aß42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. CONCLUSION: Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.
Assuntos
Perda do Osso Alveolar , Demência , Modelos Animais de Doenças , Camundongos Transgênicos , Periodontite , Ligante RANK , Animais , Feminino , Perda do Osso Alveolar/patologia , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/metabolismo , Masculino , Camundongos , Demência/etiologia , Humanos , Idoso , Ligante RANK/análise , Ligante RANK/metabolismo , Fatores Sexuais , Periodontite/complicações , Periodontite/patologia , Microtomografia por Raio-X , Osteoclastos/patologia , Peptídeos beta-Amiloides/metabolismo , Líquido do Sulco Gengival/química , Fragmentos de Peptídeos/análise , Fatores de RiscoRESUMO
Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.
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Osteoclastos , Osteócitos , Animais , Feminino , Camundongos , Cálcio/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Meios de Cultivo Condicionados/farmacologia , Osteoclastos/metabolismo , Osteócitos/metabolismo , Caracteres SexuaisRESUMO
Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2-/- and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2-/- cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk-/- mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.
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Transplante de Células-Tronco Hematopoéticas , Oxigênio , Animais , Células da Medula Óssea , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Camundongos , Camundongos Knockout , Oxigênio/farmacologiaRESUMO
The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunidade nas Mucosas , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Citocinas/sangue , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Neutralização , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Nucleocapsídeo/metabolismo , Pan troglodytes , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.
Assuntos
Adipócitos/citologia , Técnicas de Cultura de Células , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Osteócitos/citologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Fator de Crescimento de Fibroblastos 23/metabolismo , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca2+/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca2+/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein-protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database ( https://www.wikipathways.org/index.php/Pathway:WP4874 ). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.
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Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.
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The skeletal system is of paramount importance in advanced stage prostate cancer (PCa) as it is the preferred site of metastasis. Complex mechanisms are employed sequentially by PCa cells to home to and colonize the bone. Bone-resident PCa cells then recruit osteoblasts (OBs), osteoclasts (OCs), and macrophages within the niche into entities that promote cancer cell growth and survival. Since PCa is heavily reliant on androgens for growth and survival, androgen-deprivation therapy (ADT) is the standard of care for advanced disease. Although it significantly improves survival rates, ADT detrimentally affects bone health and significantly increases the risk of fractures. Moreover, whereas the majority patients with advanced PCa respond favorably to androgen deprivation, most experience a relapse of the disease to a hormone-refractory form within 1-2 years of ADT. The tumor adapts to surviving under low testosterone conditions by selecting for mutations in the androgen receptor (AR) that constitutively activate it. Thus, AR signaling remains active in PCa cells and aids in its survival under low levels of circulating androgens and additionally allows the cancer cells to manipulate the bone microenvironment to fuel its growth. Hence, AR and its downstream effectors are attractive targets for therapeutic interventions against PCa. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), was recently identified as a key downstream target of AR in coordinating PCa cell growth, survival, and migration. Additionally, this multifunctional serine/threonine protein kinase is a critical mediator of bone remodeling and macrophage function, thus emerging as an attractive therapeutic target downstream of AR in controlling metastatic PCa and preventing ADT-induced bone loss. Here, we discuss the role played by AR-CaMKK2 signaling axis in PCa survival, metabolism, cell growth, and migration as well as the cell-intrinsic roles of CaMKK2 in OBs, OCs, and macrophages within the bone microenvironment.
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Adenoid cystic carcinoma is very rare, with an incidence of 0.1 to 0.26 per one lakh people. It is the second most common primary malignancy of the trachea. The etiology, clinical manifestation and prognosis of ACC trachea in Indian scenario has not been discussed so far. We evaluated the clinical presentation, treatment and follow up details of six patients with ACC of trachea, who were treated in our Institute from January, 2006 to October,2014. Cough with expectoration, exertional dyspnoea and haemoptysis were the most frequent presentations. Proximal trachea was involved in five patients with lesion extending to subglottis in two patients. The male: female ratio was equal and most patients presented in the 4th decade of their life. Two patients were treated with total laryngectomy with proximal tracheal resection, two with tracheal resection and anastomosis and one with window resection. Four patients who were treated surgically received post operative radiation. One patient with inoperable disease was treated with radical radiotherapy. All the patients are doing well with a median follow up period of 42.8 months.
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CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Proliferação de Células , Ciclina A/metabolismo , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Imunofenotipagem , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células U937RESUMO
Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²âº/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2â»/â» mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2â»/â» mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis.
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Remodelação Óssea/fisiologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/fisiologia , Osteoblastos/enzimologia , Osteoclastos/enzimologia , Adulto , Animais , Benzimidazóis/farmacologia , Células da Medula Óssea/enzimologia , Remodelação Óssea/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Diferenciação Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/enzimologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Naftalimidas/farmacologia , Osteoblastos/citologia , Osteoclastos/citologia , Osteoporose/tratamento farmacológico , Osteoporose/enzimologia , Osteoporose/genética , OvariectomiaRESUMO
Hematopoietic stem cells (HSC) are a relatively quiescent pool of cells that perform the arduous task of replacing the short-lived mature cells of the peripheral blood. While a rapid expansion of HSCs under periods of hematological stress is warranted, their enhanced proliferation during homeostasis leads to loss of function. We recently reported that in HSCs, the evolutionarily conserved growth factor erv1-like (Gfer) acts to counter jun activation domain-binding protein 1 (Jab1)-mediated nuclear export and destabilization of the cell cycle inhibitor, p27kip1, by directly binding to and sequestering the COP9 signalosome (CSN) subunit. Through this mechanism, Gfer promotes quiescence and maintains the functional integrity of HSCs. Here, we extend our study to demonstrate an association between Gfer and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of HSC proliferation. Highly proliferative and functionally deficient Camk4-/- HSCs possess significantly lower levels of Gfer and p27kip1. Ectopic expression of Gfer restores quiescence and elevates p27kip1 expression in Camk4-/- HSCs. These results further substantiate a critical role for Gfer in the restriction of unwarranted proliferation in HSCs through the inhibition of Jab1 and subsequent stabilization and nuclear retention of p27kip1. This Gfer-mediated pro-quiescence mechanism could be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27kip1.
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Redutases do Citocromo/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Complexo do Signalossomo COP9 , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Redutases do Citocromo/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Peptídeo Hidrolases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.
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Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Complexo do Signalossomo COP9 , Sobrevivência Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação para Baixo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lentivirus , Camundongos , Camundongos Endogâmicos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Peptídeo Hidrolases/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Transfecção , Irradiação Corporal TotalRESUMO
The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.
Assuntos
Diferenciação Celular , DNA Helicases/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismo , Osteogênese , Osteopetrose/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fosfatase Ácida/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Catepsina K , Catepsinas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Isoenzimas/metabolismo , Camundongos , Camundongos Mutantes , Fator de Transcrição Associado à Microftalmia/genética , NF-kappa B/metabolismo , Osteoclastos/enzimologia , Osteogênese/genética , Osteopetrose/genética , Osteopetrose/patologia , Regiões Promotoras Genéticas , Ligante RANK/metabolismo , RNA Mensageiro/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.
Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/imunologia , Proteína de Ligação a CREB/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Baço/citologia , Baço/imunologia , Baço/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Proteína bcl-X/genética , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismoRESUMO
Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição Associado à Microftalmia/metabolismo , Células Progenitoras Mieloides/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Osteoclastos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Proteínas de Transporte/genética , Catepsina K , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Células Progenitoras Mieloides/citologia , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fosfatase Ácida Resistente a Tartarato , Transativadores/genética , Dedos de ZincoRESUMO
The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin's proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.
Assuntos
Proliferação de Células , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Adipócitos/fisiologia , Adiponectina/metabolismo , Animais , Medula Óssea/fisiologia , Células Cultivadas , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Transgênicos , Osteoblastos/fisiologia , Receptores de Adiponectina , Receptores de Superfície Celular/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure, characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn, these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Enzimológica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Homeostase , Animais , Apoptose , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proliferação de Células , Transplante de Células , Células Cultivadas , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Congênicos , Camundongos Knockout , Modelos Biológicos , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Serina/metabolismo , Fatores de Tempo , Transplante HomólogoRESUMO
UNLABELLED: The coordination of cell cycle progression and osteoclast differentiation by RANKL signaling was studied. Experiments with mouse genetic models revealed that RANKL promoted cell cycle withdrawal of osteoclast precursors dependent on the cyclin kinase inhibitor p27-KIP1, but that both p27-KIP1 and p21-CIP1 were required for osteoclast differentiation. These cyclin inhibitors may directly regulate osteoclast differentiation in addition to regulating cell cycle withdrawal. INTRODUCTION: RANKL stimulates mononuclear precursor cells of the myeloid lineage to differentiate into multinuclear osteoclasts, thus providing a system to study the fundamental problem of coordination of cell cycle progression with cell differentiation. MATERIALS AND METHODS: Mice that lack expression of functional cyclin inhibitors p27KIP1and p21CIP1 were used to study cell cycle progression and differentiation of osteoclast precursors in vitro and in vivo. RESULTS AND CONCLUSIONS: Experiments with cells derived from p27KIP1- and p21CIP1-deficient mice indicated that p27KIP1 function alone was necessary for RANKL-mediated cell cycle withdrawal by osteoclast precursors, but osteoclasts from mice with single mutations in either of these two genes differentiated normally. In contrast, p21/p27 double knockout mice developed osteopetrosis, with fewer osteoclasts that exhibited lower TRACP activity and abnormal cell morphology present in long bone. Moreover, isolated osteoclast progenitors from p21/p27 double knockout mice were defective in RANKL-mediated differentiation in vitro, expressing low levels of osteoclast-specific genes like TRACP and cathepsin K. Taken together, these data suggest p27KIP1 and p21CIP1 play roles in osteoclast differentiation in response to RANKL signaling distinct from their roles in promoting cell cycle withdrawal.