RESUMO
There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cinurenina/farmacologia , Osteogênese , Ligante RANK/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Receptores de Hidrocarboneto Arílico/genética , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Bone marrow is a reservoir for regulatory T (T(reg)) cells, but how T(reg) cells are regulated in that environment remains poorly understood. We show that expression of glucocorticoid (GC)-induced leucine zipper (GILZ) in bone marrow mesenchymal lineage cells or bone marrow-derived mesenchymal stem cells (BMSCs) increases the production of T(reg) cells via a mechanism involving the up-regulation of developmental endothelial locus-1 (Del-1), an endogenous leukocyte-endothelial adhesion inhibitor. We found that the expression of Del-1 is increased â¼4-fold in the bone tissues of GILZ transgenic (Tg) mice, and this increase is coupled with a significant increase in the production of IL-10 (2.80 vs. 0.83) and decrease in the production of IL-6 (0.80 vs. 2.33) and IL-12 (0.25 vs. 1.67). We also show that GILZ-expressing BMSCs present antigen in a way that favors T(reg) cells. These results indicate that GILZ plays a critical role mediating the crosstalk between BMSCs and T(reg) in the bone marrow microenvironment. These data, together with our previous findings that overexpression of GILZ in BMSCs antagonizes TNF-α-elicited inflammatory responses, suggest that GILZ plays important roles in bone-immune cell communication and BMSC immune suppressive functions.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células da Medula Óssea/imunologia , Comunicação Celular/imunologia , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/imunologia , Animais , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/citologia , Células da Medula Óssea/citologia , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Moléculas de Adesão Celular , Comunicação Celular/genética , Tolerância Imunológica/genética , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/citologia , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfaRESUMO
Glucocorticoids (GCs) have both anabolic and catabolic effects on bone. However, no GC anabolic effect mediator has been identified to date. Here we show that targeted expression of glucocorticoid-induced leucine zipper (GILZ), a GC anti-inflammatory effect mediator, enhances bone acquisition in mice. Transgenic mice, in which the expression of GILZ is under the control of a 3.6-kb rat type I collagen promoter, exhibited a high bone mass phenotype with significantly increased bone formation rate and osteoblast numbers. The increased osteoblast activity correlates with enhanced osteogenic differentiation and decreased adipogenic differentiation of bone marrow stromal cell cultures in vitro. In line with these changes, the mRNA levels of key osteogenic regulators (Runx2 and Osx) increased, and the level of adipogenic regulator peroxisome proliferator-activated receptor (PPAR) γ2 decreased significantly. We also found that GILZ physically interacts with C/EBPs and disrupts C/EBP-mediated PPARγ gene transcription. In conclusion, our results showed that GILZ is capable of increasing bone acquisition in vivo, and this action is mediated via a mechanism involving the inhibition of PPARγ gene transcription and shifting of bone marrow MSC/progenitor cell lineage commitment in favor of the osteoblast pathway.
Assuntos
Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica/fisiologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Fatores de Transcrição/biossíntese , Transcrição Gênica/fisiologia , Animais , Células da Medula Óssea/citologia , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas/fisiologia , Ratos , Fator de Transcrição Sp7 , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genéticaRESUMO
We had shown that aromatic amino acid (phenylalanine, tyrosine, and tryptophan) supplementation prevented bone loss in an aging C57BL/6 mice model. In vivo results from the markers of bone breakdown suggested an inhibition of osteoclastic activity or differentiation. To assess osteoclastic differentiation, we examined the effects of aromatic amino acids on early /structural markers as vitronectin receptor, calcitonin receptor, and carbonic anhydrase II as well as, late/functional differentiation markers; cathepsin K and matrix metalloproteinase 9 (MMP-9). Our data demonstrate that the aromatic amino acids down-regulated early and late osteoclastic differentiation markers as measured by real time PCR. Our data also suggest a link between the vitronectin receptor and the secreted cathepsin K that both showed consistent effects to the aromatic amino acid treatment. However, the non-attachment related proteins, calcitonin receptor, and carbonic anhydrase II, demonstrated less consistent effects in response to treatment. Our data are consistent with aromatic amino acids down-regulating osteoclastic differentiation by suppressing remodeling gene expression thus contributing initially to the net increase in bone mass seen in vivo.
Assuntos
Aminoácidos Aromáticos/farmacologia , Osteoclastos/efeitos dos fármacos , Fenilalanina/farmacologia , Triptofano/farmacologia , Tirosina/farmacologia , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Dieta , Suplementos Nutricionais , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Bone marrow skeletal stem cells (SSCs) secrete many cytokines including stromal derived factor-1 or CXCL12, which influences cell proliferation, migration, and differentiation. All CXCL12 splice variants are rapidly truncated on their N-terminus by dipeptidyl peptidase 4 (DPP4). This includes the common variant CXCL12 alpha (1-68) releasing a much less studied metabolite CXCL12(3-68). Here, we found that CXCL12(3-68) significantly inhibited SSC osteogenic differentiation and RAW-264.7 cell osteoclastogenic differentiation and induced a senescent phenotype in SSCs. Importantly, pre-incubation of SSCs with CXCL12(3-68) significantly diminished their ability to migrate toward CXCL12(1-68) in transwell migration assays. Using a high-throughput G-protein-coupled receptor (GPCR) screen (GPCRome) and bioluminescent resonance energy transfer molecular interaction assays, we revealed that CXCL12(3-68) acts via the atypical cytokine receptor 3-mediated ß-arrestin recruitment and as a competitive antagonist to CXCR4-mediated signaling. Finally, a reverse phase protein array assay revealed that DPP4-cleaved CXCL12 possesses a different downstream signaling profile from that of intact CXCL12 or controls. The data presented herein provides insights into regulation of CXCL12 signaling. Importantly, it demonstrates that DPP4 proteolysis of CXCL12 generates a metabolite with significantly different and previously overlooked bioactivity that helps explain discrepancies in the literature. This also contributes to an understanding of the molecular mechanisms of osteoporosis and bone fracture repair and could potentially significantly affect the interpretation of experimental outcomes with clinical consequences in other fields where CXCL12 is vital, including cancer biology, immunology, cardiovascular biology, neurobiology, and associated pathologies.
RESUMO
Although the gut microbiota has been reported to influence osteoporosis risk, the individual species involved, and underlying mechanisms, remain largely unknown. We performed integrative analyses in a Chinese cohort of peri-/post-menopausal women with metagenomics/targeted metabolomics/whole-genome sequencing to identify novel microbiome-related biomarkers for bone health. Bacteroides vulgatus was found to be negatively associated with bone mineral density (BMD), which was validated in US white people. Serum valeric acid (VA), a microbiota derived metabolite, was positively associated with BMD and causally downregulated by B. vulgatus. Ovariectomized mice fed B. vulgatus demonstrated increased bone resorption and poorer bone micro-structure, while those fed VA demonstrated reduced bone resorption and better bone micro-structure. VA suppressed RELA protein production (pro-inflammatory), and enhanced IL10 mRNA expression (anti-inflammatory), leading to suppressed maturation of osteoclast-like cells and enhanced maturation of osteoblasts in vitro. The findings suggest that B. vulgatus and VA may represent promising targets for osteoporosis prevention/treatment.
Assuntos
Reabsorção Óssea , Microbioma Gastrointestinal , Osteoporose , Humanos , Feminino , Camundongos , AnimaisRESUMO
Matrix metalloproteinase-1 (MMP-1) is a member of the family of zinc-dependent endopeptidases that are capable of degrading extracellular matrix (ECM) and certain non-matrix proteins. It has been shown that MMP-1 can enhance muscle regeneration by improving the differentiation and migration of myoblasts. However, it is still not known whether MMP-1 can promote the myogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). To address this question, we isolated BMSCs from C57BL/6J mice and investigated the effects of MMP-1 on their proliferation and myogenic differentiation. Our results showed that MMP-1 treatment, which had no cytotoxic effects on BMSCs, increased the mRNA and protein levels of MyoD and desmin in a dose-dependent manner, indicating that MMP-1 promoted myogenic differentiation of BMSCs in vitro. These results suggest that BMSCs may have a therapeutic potential for treating muscular disorders.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Metaloproteinase 1 da Matriz/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Separação Celular , Células Cultivadas , Desmina/biossíntese , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/biossíntese , Osteogênese/efeitos dos fármacosRESUMO
Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 µM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated ß-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3',4'-dimethoxyflavone to try to block AhR signaling and rescue kynurenine /AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated ß-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis.
Assuntos
Autofagia/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cinurenina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Osteoporose , Transdução de Sinais , beta-Galactosidase/efeitos dos fármacosRESUMO
Tryptophan is an essential amino acid catabolized initially to kynurenine (kyn), an immunomodulatory metabolite that we have previously shown to promote bone loss. Kyn levels increase with aging and have also been associated with neurodegenerative disorders. Picolinic acid (PA) is another tryptophan metabolite downstream of kyn. However, in contrast to kyn, PA is reported to be neuroprotective and further, to promote osteogenesis in vitro. Thus, we hypothesized that PA might be osteoprotective in vivo. In an IACUC-approved protocol, we fed PA to aged (23-month-old) C57BL/6 mice for eight weeks. In an effort to determine potential interactions of PA with dietary protein we also fed PA in a low-protein diet (8%). The mice were divided into four groups: Control (18% dietary protein), +PA (700 ppm); Low-protein (8%), +PA (700 ppm). The PA feedings had no impact on mouse weight, body composition or bone density. At sacrifice bone and stem cells were collected for analysis, including µCT and RT-qPCR. Addition of PA to the diet had no impact on trabecular bone parameters. However, marrow adiposity was significantly increased in PA-fed mice, and in bone marrow stromal cells isolated from these mice increases in the expression of the lipid storage genes, Plin1 and Cidec, were observed. Thus, as a downstream metabolite of kyn, PA no longer showed kyn's detrimental effects on bone but instead appears to impact energy balance.
Assuntos
Adiposidade , Triptofano , Animais , Densidade Óssea , Medula Óssea , Camundongos , Camundongos Endogâmicos C57BL , Ácidos PicolínicosRESUMO
Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.
RESUMO
Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphoma in poultry. The complete genome of the avirulent vaccine strain MDV-814 was cloned as an infectious bacterial artificial chromosome (BAC) using an 8.8-kb fragment containing the self-designed selective marker guanosine phosphoriboxyl transferase. The recombinant virus MDV-814-BAC was generated by co-transfection of a BAC transfer vector and MDV-814 total DNA, and was purified by eight rounds of selective passaging. The infectivity of the BAC DNA clone was validated by MDV reconstitution from chicken embryo fibroblasts transfected with MDV-BAC DNA, which was extracted from electroporated Escherichia coli DH10B cells. In vitro, the BAC-derived virus had similar biological characteristics and growth kinetics as the wild-type parental and recombinant viruses, and chickens immunized with BAC derivatives by various delivery mechanisms acquired protection against virulent MDV challenge. Construction of this MDV-BAC may aid the development of recombinant vaccines-containing multiple antigens.
Assuntos
Galinhas/virologia , Cromossomos Artificiais Bacterianos/genética , Mardivirus/genética , Vacinas contra Doença de Marek/genética , Doença de Marek/prevenção & controle , Animais , Galinhas/imunologia , Clonagem Molecular , DNA Viral/genética , Vetores Genéticos , Genoma Viral , Mardivirus/crescimento & desenvolvimento , Mardivirus/imunologia , Doença de Marek/imunologia , Doença de Marek/virologia , Vacinas contra Doença de Marek/imunologia , Vacinas de DNA/genéticaRESUMO
The aim of this study was to elucidate the concise effects of a traditional herb pair, Curcumae rhizoma-Sparganii rhizoma (CRSR), on uterine leiomyoma (UL) by analyzing transcriptional profiling. The UL rat model was made by intramuscular injection of progesterone and gavage administration of diethylstilbestrol. From 11 weeks of the establishment of the model, rats of the UL+CRSR group were gavaged daily with CRSR (6.67 g/kg). The serum concentrations of progesterone (P) and estradiol (E2) were determined by radioimmunoassay, the uterine index was measured by caliper measurement, and the pathological status was observed by hematoxylin and eosin stain. Gene expression profiling was checked by NimbleGen Rat Gene Expression Microarrays. The results indicated that the uterine mass of UL+CRSR rats was significantly shrunk and serum P and E2 levels significantly reduced compared to UL animals and nearly to the level of normal rats. Results of microarrays displayed the extensive inhibition of CRSR upon the expression of proliferation and deposition of extracellular matrix (ECM)-related genes, and significantly regulated a wide range of metabolism disorders. Furthermore, CRSR extensively regulated key pathways of the UL process, such as MAPK, PPAR, Notch, and TGF-ß/Smad. Regulation of the crucial pathways for the UL process and ECM metabolism may be the underlying mechanisms of CRSR treatment. Further studies will provide clear clues for effectively treating UL with CRSR.
Assuntos
Curcuma/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leiomioma/tratamento farmacológico , Extratos Vegetais/farmacologia , Rizoma/química , Neoplasias Uterinas/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Leiomioma/genética , Leiomioma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
Cyclooxygenase-2 (COX-2) plays an important role in rheumatoid arthritis and therefore, has been a major target for anti-arthritis therapies. The expression of COX-2 is induced by inflammatory cytokines such as TNF-alpha and IL-1beta, and inhibited by glucocorticoids. However, the molecular mechanisms underlying the anti-inflammatory and immune suppressive actions of glucocorticoids are not well defined. Here we report that glucocorticoid-induced leucine zipper (GILZ) mimics glucocorticoid action and inhibits inflammatory cytokine-induced COX-2 expression in bone marrow mesenchymal stem cells, the cells that have been recently implicated in the pathogenesis and progression of rheumatoid arthritis. Using a retrovirus-mediated gene expression approach we demonstrate that overexpression of GILZ inhibits TNF-alpha and IL-1beta-induced COX-2 mRNA and protein expression, and knockdown of GILZ by shRNA reduces glucocorticoid inhibition of cytokine-induced COX-2 expression. Consistent to these results, overexpression of GILZ also inhibits NF-kappaB-mediated COX-2 promoter activity. Finally, we show that GILZ inhibits COX-2 expression by blocking NF-kappaB nuclear translocation. Our results suggest that GILZ is a key glucocorticoid effect mediator and that GILZ may have therapeutic value for novel anti-inflammation therapies.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Glucocorticoides/fisiologia , Interleucina-1beta/fisiologia , Células-Tronco Mesenquimais/metabolismo , Fatores de Transcrição/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/genética , Regulação da Expressão Gênica , Glucocorticoides/farmacologia , Zíper de Leucina , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis. It is also a central player in energy metabolism, inflammation and immunity. As an important nuclear transcription factor, PPARγ can regulate the expression and function of genes or biological processes directly or indirectly via association with other factors and thus modulate their activities. To better understand the impact of PPARγ on the global gene expression profile, we evaluated the bioinformatic data, which revealed the changes that occurred in genes and their pathways in the absence of PPARγ. In brief, we performed RNA deep sequencing (RNA-Seq) analysis using RNA samples isolated from multipotent mesenchymal stromal cells (MSCs) of PPARγ knockout and wild type control mice. The RNA-Seq data sets were then subjected to bioinformatic analyses from various angles to better reveal the breadth of PPARγ function in different biological processes. Our results reveal novel genes and networks modulated by PPARγ and provides new insights into our understanding of the physiologic and pathophysiologic role this nuclear receptor plays in health and disease.
Assuntos
Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , PPAR gama/metabolismo , Animais , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Camundongos Knockout , Reprodutibilidade dos TestesRESUMO
Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.
Assuntos
Densidade Óssea , Deleção de Genes , Osteogênese , Proteína Quinase C/genética , Células-Tronco/enzimologia , Adenoviridae/metabolismo , Fosfatase Alcalina/metabolismo , Aminoácidos/sangue , Animais , Células da Medula Óssea/citologia , Calcificação Fisiológica , Integrases/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Transgênicos , Proteína Quinase C/metabolismoRESUMO
TNF-α plays a key role in the development of rheumatoid arthritis (RA) and inflammatory bone loss. Unfortunately, treatment of RA with anti-inflammatory glucocorticoids (GCs) also causes bone loss resulting in osteoporosis. Our previous studies showed that overexpression of glucocorticoid-induced leucine zipper (GILZ), a mediator of GC's anti-inflammatory effect, can enhance osteogenic differentiation in vitro and bone acquisition in vivo. To investigate whether GILZ could antagonize TNF-α-induced arthritic inflammation and protect bone in mice, we generated a TNF-α-GILZ double transgenic mouse line (TNF-GILZ Tg) by crossbreeding a TNF-α Tg mouse, which ubiquitously expresses human TNF-α, with a GILZ Tg mouse, which expresses mouse GILZ under the control of a 3.6kb rat type I collagen promoter fragment. Results showed that overexpression of GILZ in bone marrow mesenchymal stem/progenitor cells protected mice from TNF-α-induced inflammatory bone loss and improved bone integrity (TNF-GILZ double Tg vs. TNF-αTg, n = 12-15). However, mesenchymal cell lineage restricted GILZ expression had limited effects on TNF-α-induced arthritic inflammation as indicated by clinical scores and serum levels of inflammatory cytokines and chemokines.
Assuntos
Artrite/metabolismo , Artrite/patologia , Fêmur/patologia , Fatores de Transcrição/metabolismo , Animais , Artrite/genética , Artrite/fisiopatologia , Densidade Óssea , Quimiocinas/sangue , Fêmur/fisiopatologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. µCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.
Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Reabsorção Óssea/patologia , Cinurenina/metabolismo , Triptofano/metabolismo , Adiposidade , Envelhecimento/sangue , Animais , Peso Corporal , Reabsorção Óssea/sangue , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/metabolismo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Calcificação Fisiológica , Diferenciação Celular , Dieta , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/análogos & derivados , Cinurenina/sangue , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Microtomografia por Raio-XRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ), known as the master regulator of adipogenesis, has been regarded as a promising target for new anti-osteoporosis therapy due to its role in regulating bone marrow mesenchymal stem/progenitor cell (BMSC) lineage commitment. However, the precise mechanism underlying PPARγ regulation of bone is not clear as a bone-specific PPARγ conditional knockout (cKO) study has not been conducted and evidence showed that deletion of PPARγ in other tissues also have profound effect on bone. In this study, we show that mice deficiency of PPARγ in cells expressing a 3.6 kb type I collagen promoter fragment (PPAR(fl/fl):Col3.6-Cre) exhibits a moderate, site-dependent bone mass phenotype. In vitro studies showed that adipogenesis is abolished completely and osteoblastogenesis increased significantly in both primary bone marrow culture and the BMSCs isolated from PPARγ cKO mice. Histology and histomorphometry studies revealed significant increases in the numbers of osteoblasts and surface in the PPARγ cKO mice. Finally, we found that neither the differentiation nor the function of osteoclasts was affected in the PPARγ cKO mice. Together, our studies indicate that PPARγ plays an important role in bone remodeling by increasing the abundance of osteoblasts for repair, but not during skeletal development.
Assuntos
Adipogenia , Remodelação Óssea , Osteogênese , PPAR gama/genética , PPAR gama/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Inativação de Genes , Células-Tronco Mesenquimais/fisiologia , Camundongos , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Regiões Promotoras GenéticasRESUMO
Age-induced bone loss is associated with greater bone resorption and decreased bone formation resulting in osteoporosis and osteoporosis-related fractures. The etiology of this age-induced bone loss is not clear but has been associated with increased generation of reactive oxygen species (ROS) from leaky mitochondria. ROS are known to oxidize/damage the surrounding proteins/amino acids/enzymes and thus impair their normal function. Among the amino acids, the aromatic amino acids are particularly prone to modification by oxidation. Since impaired osteoblastic differentiation from bone marrow mesenchymal stem cells (BMMSCs) plays a role in age-related bone loss, we wished to examine whether oxidized amino acids (in particular the aromatic amino acids) modulated BMMSC function. Using mouse BMMSCs, we examined the effects of the oxidized amino acids di-tyrosine and kynurenine on proliferation, differentiation and Mitogen-Activated Protein Kinase (MAPK) pathway. Our data demonstrate that amino acid oxides (in particular kynurenine) inhibited BMMSC proliferation, alkaline phosphatase expression and activity and the expression of osteogenic markers (Osteocalcin and Runx2). Taken together, our data are consistent with a potential pathogenic role for oxidized amino acids in age-induced bone loss.
Assuntos
Aminoácidos Aromáticos/farmacologia , Anabolizantes/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoporose/etiologia , Oxirredução , Aminoácidos Aromáticos/química , Anabolizantes/química , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Triptofano/química , Triptofano/farmacologia , Tirosina/química , Tirosina/farmacologiaRESUMO
Glucocorticoids are the only therapy that has been demonstrated to alter the progress of Duchenne muscular dystrophy (DMD), the most common muscular dystrophy in children. However, glucocorticoids disturb skeletal muscle metabolism and hamper myogenesis and muscle regeneration. The mechanisms involved in the glucocorticoid-mediated suppression of myogenic differentiation are not fully understood. Glycogen synthase kinase-3ß (GSK-3ß) is considered to play a central role as a negative regulator in myogenic differentiation. Here, we showed that glucocorticoid treatment during the first 48 h in differentiation medium decreased the level of phosphorylated Ser9-GSK-3ß, an inactive form of GSK-3ß, suggesting that glucocorticoids affect GSK-3ß activity. We then investigated whether GSK-3ß inhibition could regulate glucocorticoid-mediated suppression of myogenic differentiation in vitro. Two methods were employed to inhibit GSK-3ß: pharmacological inhibition with LiCl and GSK-3ß gene knockdown. We found that both methods resulted in enhanced myotube formation and increased levels of muscle regulatory factors and muscle-specific protein expression. Importantly, GSK-3ß inhibition attenuated glucocorticoid-induced suppression of myogenic differentiation. Collectively, these data suggest the involvement of GSK-3ß in the glucocorticoid-mediated impairment of myogenic differentiation. Therefore, the inhibition of GSK-3ß may be a strategy for preventing glucocorticoid-induced muscle degeneration.