ABSTRACT
Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.
Subject(s)
Bone Marrow Diseases , Exocrine Pancreatic Insufficiency , Lipomatosis , Humans , Shwachman-Diamond Syndrome , Tumor Suppressor Protein p53/genetics , Lipomatosis/genetics , Codon, Nonsense , Myelopoiesis , Neutrophils/metabolism , Chemotaxis , Bone Marrow Diseases/genetics , Bone Marrow Diseases/therapy , Exocrine Pancreatic Insufficiency/genetics , Ribosomes/metabolismABSTRACT
Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected ß-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. We report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in ß-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.
Subject(s)
Bone Marrow/pathology , Hematopoietic Stem Cells/pathology , Stem Cell Niche , beta-Thalassemia/pathology , Animals , Female , Hematopoiesis/physiology , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.
Subject(s)
Calcification, Physiologic , Osteoblasts/metabolism , Shwachman-Diamond Syndrome/metabolism , Tumor Suppressor Protein p53/metabolism , Cells, Cultured , Female , Humans , Male , Osteoblasts/pathology , Proteins/genetics , Proteins/metabolism , Shwachman-Diamond Syndrome/genetics , Shwachman-Diamond Syndrome/pathology , Tumor Suppressor Protein p53/geneticsABSTRACT
Growth hormone (GH) is best known for its prominent role in promoting prepubertal growth and in regulating body composition and metabolism during adulthood. In recent years, the possible role of GH in the modulation of mesenchymal stem cell (MSC) commitment has gained interest. MSCs, characterized by active self-renewal and differentiation potential, express GH receptors. In MSCs derived from different adult tissues, GH induces an inhibition of adipogenic differentiation and favors MSC differentiation towards osteogenesis. This activity of GH indicates that regulation of body composition by GH has already started in the tissue progenitor cells. These findings have fostered research on possible uses of MSCs treated with GH in those pathologies, where a lack of or delays in bone repair occur. After an overview of GH activities, this review will focus on the research that has characterized GH's effects on MSCs and on preliminary studies on the possible application of GH in bone regenerative medicine.
Subject(s)
Bone Regeneration , Growth Hormone/metabolism , Mesenchymal Stem Cells/metabolism , Animals , Cell Differentiation , Humans , Mesenchymal Stem Cells/cytology , Regenerative Medicine/methodsABSTRACT
BACKGROUND: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. METHODS: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. RESULTS: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. CONCLUSIONS: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality.
Subject(s)
Betaine/pharmacology , Cell Differentiation/drug effects , Osteoblasts/cytology , Aged , Aged, 80 and over , Calcium/metabolism , Cells, Cultured , Gene Expression Regulation/drug effects , Humans , Membrane Potentials/drug effects , Models, Biological , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteogenesis/drug effects , Osteogenesis/genetics , Signal Transduction/drug effects , Superoxide Dismutase/metabolismABSTRACT
The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.
Subject(s)
Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis Imperfecta/metabolism , Adipogenesis/drug effects , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Autophagy/drug effects , Blotting, Western , Boronic Acids/pharmacology , Bortezomib , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Flow Cytometry , Immunohistochemistry , Mice , Osteogenesis/drug effects , Osteogenesis Imperfecta/pathology , Pyrazines/pharmacologyABSTRACT
Clinical evidence highlights a relationship between the blood and the bone, but the underlying mechanism linking these two tissues is not fully elucidated. Here, we used ß-thalassemia as a model of congenital anemia with bone and bone marrow (BM) niche defects. We demonstrate that fibroblast growth factor 23 (FGF23) is increased in patients and mice with ß-thalassemia because erythropoietin induces FGF23 overproduction in bone and BM erythroid cells via ERK1/2 and STAT5 pathways. We show that in vivo inhibition of FGF23 signaling by carboxyl-terminal FGF23 peptide is a safe and efficacious therapeutic strategy to rescue bone mineralization and deposition in mice with ß-thalassemia, normalizing the expression of niche factors and restoring hematopoietic stem cell (HSC) function. FGF23 may thus represent a molecular link connecting anemia, bone, and the HSC niche. This study provides a translational approach to targeting bone defects and rescuing HSC niche interactions, with potential clinical relevance for improving HSC transplantation and gene therapy for hematopoietic disorders.
Subject(s)
Hematopoietic Stem Cell Transplantation , beta-Thalassemia , Animals , Mice , beta-Thalassemia/therapy , Bone Marrow , Bone Marrow Cells/metabolism , Hematopoietic Stem Cells/metabolism , Stem Cell Niche , HumansABSTRACT
Though extensive studies have been conducted, questions regarding the molecular effectors and pathways underlying the regulatory role of 1,25(OH)(2)D(3) in human osteoblasts other than cell differentiation and matrix protein production remain unanswered. This study aims to identify genes and pathways that are modulated by 1,25(OH)(2)D(3) treatment in human osteoblasts. Primary osteoblast cultures obtained from human bone tissue samples were treated with 1,25(OH)(2)D(3) (10(-7) M) for 24 h and their transcritptomes were profiled by microarray analysis using the Affymetrix GeneChip. Statistical analysis was conducted to identify genes whose expression is significantly modulated following 1,25(OH)(2)D(3) treatment. One hundred and fifty-eight genes were found to be differentially expressed. Of these, 136 were upregulated, indicating clear transcriptional activation by 1,25(OH)(2)D(3). Biostatistical evaluation of microarray data by Ingenuity Pathways Analysis (IPA) revealed a relevant modulation of genes involved in vitamin D metabolism (CYP24), immune functions (CD14), neurotransmitter transporters (SLC1A1, SLC22A3), and coagulation [thrombomodulin (THBD), tissue plasminogen activator (PLAT), endothelial protein C receptor (PROCR), thrombin receptor (F2R)]. We identified a restricted number of highly regulated genes and confirmed their differential expression by real-time quantitative PCR (RT qPCR). The present genome-wide microarray analysis on 1,25(OH)(2)D(3) -treated human osteoblasts reveals an interplay of critical regulatory and metabolic pathways and supports the hypothesis that 1,25(OH)(2)D(3) can modulate the coagulation process through osteoblasts, activates osteoclastogenesis through inflammation signaling, modulates the effects of monoamines by affecting their reuptake.
Subject(s)
Calcitriol/pharmacology , Gene Expression Regulation , Osteoblasts/metabolism , Vitamins/pharmacology , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Differentiation , Cells, Cultured , Gene Expression Profiling , Gene Regulatory Networks , Humans , Oligonucleotide Array Sequence Analysis , Primary Cell Culture , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Bone remodeling replaces old and damaged bone with new bone through a sequence of cellular events occurring on the same surface without any change in bone shape. It was initially thought that the basic multicellular unit (BMU) responsible for bone remodeling consists of osteoclasts and osteoblasts functioning through a hierarchical sequence of events organized into distinct stages. However, recent discoveries have indicated that all bone cells participate in BMU formation by interacting both simultaneously and at different differentiation stages with their progenitors, other cells, and bone matrix constituents. Therefore, bone remodeling is currently considered a physiological outcome of continuous cellular operational processes optimized to confer a survival advantage. Bone remodeling defines the primary activities that BMUs need to perform to renew successfully bone structural units. Hence, this review summarizes the current understanding of bone remodeling and future research directions with the aim of providing a clinically relevant biological background with which to identify targets for therapeutic strategies in osteoporosis.
ABSTRACT
A link between hypertension and long-term bone health has been suggested. The aim of this study was to investigate the effects of chronic angiotensin II administration on urinary calcium/phosphate excretion, bone mineral density, bone remodeling and osteoblast population in a well-established experimental model of hypertension, in the absence of possible confounding factors that could affect bone metabolism. Male Sprague-Dawley rats, divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis, n = 8); (b) Ang II+losartan (Los, 50 mg/kg/day, per os, n = 6); (c) control group (physiological saline, sub cutis, n = 9); and (d) control+losartan (n = 6) were treated for four weeks. During the experimental period, 24-hour diuresis, urinary calcium, phosphate and sodium excretion were measured prior to the treatment, at two weeks of treatment, and at the end of the treatment. Systolic blood pressure was measured by plethysmography technique (tail cuff method). At the end of the experimental protocol, the rats were euthanized and peripheral quantitative computed tomography at the proximal metaphysis and at the diaphysis of the tibiae and quantitative bone histomorphometry on distal femora were performed. Angiotensin II-dependent hypertension is associated with increased calcium and phosphate excretion. AT1 receptor blockade prevented the increase of blood pressure and phosphate excretion but did not affect the increase of calcium excretion. These changes took place without significantly affecting bone density, bone histology or osteoblast population. In conclusion, in our experimental conditions, angiotensin II-dependent hypertension gave rise to an increased urinary excretion of calcium and phosphate without affecting bone density.
ABSTRACT
Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.
Subject(s)
Aging/physiology , Bone Marrow Transplantation/methods , Fetal Research , Osteogenesis Imperfecta/prevention & control , Osteogenesis Imperfecta/therapy , Uterus/physiology , Animals , Bone Marrow Cells/cytology , Collagen/metabolism , Disease Models, Animal , Extracellular Space/chemistry , Female , Gene Knock-In Techniques , Genes, Dominant , Graft Survival , Mice , Mice, Transgenic , Osteogenesis Imperfecta/metabolism , Osteogenesis Imperfecta/pathology , Phenotype , Pregnancy , Spectrum Analysis, Raman , Survival Rate , Tissue DonorsABSTRACT
Growth hormone (GH) replacement in adulthood results in variable bone responses as a function of the gonadic hormonal milieu. We performed a retrospective analysis of a large cohort of adult males and females with confirmed GH deficiency (GHD) prior to treatment and during 3 years of replacement therapy. Potential confounders and effect modifiers were taken into account. Sixty-four adult patients with GHD (20 females and 44 males; mean age 34 years, range 18-64) were included in the analysis. GH replacement induced a different effect on bone in males compared to females. Bone mineral content increased in males and decreased in females at the lumbar spine, total femur, and femoral neck; bone mineral density showed a similar trend at the lumbar spine and femoral neck. There was no significant gender difference in bone area at any measured bone site. In both sexes we observed a similar trend for serum markers of bone remodeling. Sex predicted bone outcome on multivariate analysis, as did age, onset of GHD (childhood/adulthood), pretreatment bone mass, baseline body mass index (BMI), and BMI change during GH replacement. Serum IGF-I levels during treatment did not show any relationship with bone outcome at any measured site. This study confirms that bone responsiveness to GH replacement in adult GHD varies as a function of sex even after controlling for potential confounders and highlights the importance of other cofactors that may affect the interaction between GH replacement therapy and bone remodeling.
Subject(s)
Hormone Replacement Therapy/methods , Human Growth Hormone/deficiency , Human Growth Hormone/therapeutic use , Absorptiometry, Photon/methods , Adolescent , Adult , Body Composition , Body Mass Index , Bone Remodeling , Bone and Bones/metabolism , Bone and Bones/physiology , Female , Humans , Male , Middle Aged , Multivariate Analysis , Sex FactorsABSTRACT
AIM: Since its discovery Prostate Specific Antigen (PSA), also referred to as kallikrein-3 (KLK3), has been used as standard circulating biomarker for prostate cancer (PCa). However, its specificity remains not adequate and its mechanism of action still elusive. Therefore, deciphering PSA role throughout PCa-pathobiology would be relevant in improving both cancer diagnosis and outcome prediction. We investigated the possible role played by PSA on/in the tumor microenvironment and over the first steps of cancer invasion. METHODS: Fresh PCa-specimens and cell lines were used for ex-vivo/in-vitro invasion assays and assessment of prostate tissue-PSA (tPSA), type 1 collagen (COL1A1) and ß1-integrin expression. Tissue Cancer Genome Atlas (TCGA) and Decipher® datasets were considered to estimate tPSA clinical relevance. RESULTS: A more precise, inverse, correspondence between tPSA and clinical/pathological parameters was found than for circulating PSA. KLK3 combined with Gleason grade and pathologic stage, better predicted cancer-related mortality. Consistently, we demonstrated that PSA inhibits prostate extracellular-matrix (ECM) invasion by PCa cells. As for the mechanism of action, we provided novel information that PSA is able to cleave COL1A1, a main component of the ECM. Finally, ß1-integrin, a crucial COL1A1 transducing-receptor involved in tumor adhesion/invasion, resulted to be downregulated in PCa specimens with higher levels of tPSA. CONCLUSIONS: By interfering with type 1 collagen and its downstream targets, PSA may hamper adhesion and path of the cancer cells through ECM and their migration ability, thus explaining the inverse correlation highlighted between prostate tPSA levels and clinically significant disease.
ABSTRACT
Transgenic mice over-expressing calcitonin gene-related peptide (CGRP) in osteoblasts have increased bone density due to increased bone formation, thus suggesting that CGRP plays a role in bone metabolism. In this study we determined the relationship between CGRP, the canonical Wnt signaling and apoptosis in human osteoblasts (hOBs) in consideration of the well-documented involvement of this pathway in bone cells. Primary cultures of hOBs were treated with CGRP 10(-8) M. Levels of ß-catenin, which is the cytoplasmic protein mediator of canonical Wnt signaling, and mRNA were determined. CGRP increases both the expression and the levels of cytoplasmic ß-catenin by binding to its receptor, as this effect is blocked by the antagonist CGRP(8-37). This facilitatory action on ß-catenin appears to be mediated by the inhibition of the enzyme GSK-3ß via protein kinase A (PKA) activation. GSK-3ß is a glycogen synthase kinase that, by phosphorylating ß-catenin, promotes its degradation by the proteosomal machinery. Moreover, the peptide is able to inhibit hOBs apoptosis stimulated by dexamethasone or by serum deprivation, possibly through the accumulation of ß-catenin, since the inhibitor of PKA activity H89 partially prevents the antiapoptotic effect of the peptide. In conclusion CGRP, released by nerve fibers, exerts its anabolic action on bone cells by stimulating canonical Wnt signaling and by inhibiting hOBs apoptosis, thus favoring local bone regeneration.
Subject(s)
Apoptosis , Bone Regeneration , Calcitonin Gene-Related Peptide/metabolism , Osteoblasts/metabolism , Peptide Fragments/metabolism , beta Catenin/metabolism , Active Transport, Cell Nucleus , Aged , Apoptosis/drug effects , Bone Regeneration/drug effects , Cell Survival , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Dexamethasone/pharmacology , Enzyme Activators/pharmacology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Isoquinolines/pharmacology , Middle Aged , Osteoblasts/drug effects , Osteoblasts/pathology , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Stability , RNA, Messenger/metabolism , Signal Transduction , Sulfonamides/pharmacology , Time Factors , Wnt Proteins/metabolism , beta Catenin/geneticsABSTRACT
Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.
Subject(s)
Bone Matrix/drug effects , Carnitine/pharmacology , Cell Differentiation/drug effects , Osteoblasts/drug effects , Oxidative Stress/drug effects , Aged , Aged, 80 and over , Antioxidants/metabolism , Bone Matrix/metabolism , Calcification, Physiologic/drug effects , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Female , Humans , Integrin-Binding Sialoprotein/metabolism , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinases/metabolism , Osteoblasts/metabolism , Osteogenesis/drug effects , Osteopontin/metabolism , Oxidation-Reduction , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , Sp7 Transcription Factor/metabolism , Up-Regulation/drug effectsABSTRACT
The imbalance between osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. In the present study, using primary human mesenchymal stromal cells (hMSCs) isolated from trabecular bone, we assessed the possible effect of GH on hMSC differentiation potential into adipocytes. GH (5â¯ng/ml) significantly inhibited the lipid accumulation in hMSCs cultured for 14â¯days in lipogenic medium. GH decreased the expression of the adipogenic genes, CCAAT/enhancer-binding protein alpha (C/EBPα) and adiponectin (ADN) as well as the expression of two lipogenesis-related enzymes, lipoprotein lipase (LPL) and acethylCoA carboxylase (ACACA). In parallel, GH induced an increase in the gene expression and protein levels of osterix (OSX) and osteoprotegerin (OPG). These effects were ascribed to enhanced Wnt signaling as GH significantly reduced Wnt inhibitors, Dickkopf 1 (DKK1) and the secreted frizzled protein 2 (SFRP2), and increased the expression of an activator of Wnt, Wnt3. Accordingly, the expression of ß-catenin and its nuclear levels were raised. Wnt involvement in GH anti-adipogenic effect was further confirmed by the silencing of ß-catenin. In silenced hMSC, both the inhibitory effect of GH on the expression of the adipogenic genes, ADN and C/EBPα and the lipogenesis enzymes LPL and ACACA, were prevented together with the stimulatory effect of GH on the osteogenic genes OSX and OPG. The present study supports the hypothesis that when GH secretion declines as in aging, the fat in the bone-marrow cavities increases and the osteogenic capacity of the MSC pool is reduced due to a decrease in Wnt signaling.
Subject(s)
Adipogenesis/drug effects , Cancellous Bone/cytology , Growth Hormone/pharmacology , Mesenchymal Stem Cells/cytology , Wnt Signaling Pathway , Adipogenesis/genetics , Gene Expression Regulation/drug effects , Gene Silencing/drug effects , Humans , Lipid Droplets/drug effects , Lipid Droplets/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteogenesis/drug effects , Osteogenesis/genetics , Wnt Signaling Pathway/drug effects , beta Catenin/metabolismABSTRACT
OBJECTIVE: Some extra-thyroid actions of thyroid stimulating hormone (TSH), such as an in vitro action on bone, have been described. Our aim was to evaluate in vivo the acute effect of a recombinant human TSH (rhTSH)-induced TSH surge on Osteoprotegerin (OPG) and receptor activator of the nuclear factor-kappaBeta (RANK-L) levels in patients under levo-thyroxine (L-T4) therapy. DESIGN: 24 patients with differentiated thyroid carcinoma (DTC) were studied. Standard rhTSH testing was performed. OPG, RANK-L, TSH, thyroid hormones, thyroglobulin and several parameters of bone metabolism were evaluated. RESULTS: Baseline OPG and RANK-L levels were in the range of our reference population. An inverse correlation between OPG and spinal Z-score (p=0.029) and between RANK-L and age (p=0.018) or urinary calcium/creatinine ratio (p=0.011) was detected. After rhTSH administration, a significant (p<0.001) increase in TSH was found. No significant increase in OPG or RANK-L levels after rhTSH was observed. No correlation was detected between TSH peak value after rhTSH and maximal percentage change in OPG or RANK-L. A slight increase in urinary cross-links after rhTSH was found. CONCLUSIONS: In a small group of subjects with a history of DTC on L-T4 regimen, our study did not support an acute direct effect of TSH on OPG and RANK-L.
Subject(s)
Osteoprotegerin/blood , RANK Ligand/blood , Thyroid Neoplasms/blood , Thyrotropin/administration & dosage , Adult , Aged , Aging , Calcium/urine , Creatinine/urine , Female , Humans , Hydroxyproline/urine , Iodine Radioisotopes/therapeutic use , Male , Middle Aged , Osteoporosis/physiopathology , Postmenopause , Premenopause , Recombinant Proteins/administration & dosage , Thyroid Neoplasms/therapy , Thyroidectomy , Thyrotropin/blood , Thyroxine/administration & dosage , Thyroxine/therapeutic useABSTRACT
Wnt signaling, a major regulator of bone formation and homeostasis, might be involved in the bone loss of osteoporotic patients and the consequent impaired response to fracture. Therefore we analyzed Wnt-related, osteogenic, and adipogenic genes in bone tissue of elderly postmenopausal women undergoing hip replacement for either femoral fracture or osteoarthritis. Bone specimens derived from the intertrochanteric region of the femurs of 25 women with fracture (F) and 29 with osteoarthritis without fracture (OA) were analyzed. Specific miRNAs were analyzed in bone and in matched blood samples. RUNX2, BGP, and OPG showed lower expression in F than in OA samples, while OSX, OPN, BSP, and RANKL were not different. Inhibitory genes of Wnt pathway were lower in F versus OA. ß-Catenin protein levels were higher in F versus OA, whereas its cotranscriptional regulator (Lef1) was lower in F group. miR-204, which targets RUNX2, and miR-130a, which inhibits PPARγ, were lower and higher, respectively, in F versus OA serum samples. The present study showed an inefficient Wnt signal transduction in F group despite higher ß-catenin protein levels, consistent with the expected overall postfracture systemic activation towards osteogenesis. This transcriptional inefficiency could contribute to the osteoporotic bone fragility.
Subject(s)
Femoral Fractures/blood , Postmenopause/blood , Wnt Signaling Pathway , Aged , Aged, 80 and over , Core Binding Factor Alpha 1 Subunit/blood , Female , Femoral Fractures/pathology , Humans , MicroRNAs/blood , Osteoarthritis/blood , Osteoarthritis/pathology , Osteoprotegerin/blood , RANK Ligand/blood , beta Catenin/bloodABSTRACT
AIM: To assess serum levels of the plaque calcification regulators osteoprotegerin (OPG) and Matrix Gla-proteins (MGP) in individuals with stable angina and acute myocardial infarction submitted to coronary angiography and their relation to coronary artery disease burden. METHODS: The study included 40 individuals affected by ST-elevation myocardial infarction (STEMI) and 40 individuals with stable angina who all underwent coronary angiography, with evaluation of the extent of coronary artery disease by Syntax Score calculation and measurement of serum OPG and MGP levels. Osteoporosis was excluded by femoral and vertebral computerized bone mineralometry. RESULTS: Serum OPG and MGP levels were respectively 3.87â±â1.07âpmol/l and 6.80â±â2.43ânmol/l in the stable angina group, 7.57â±â1.5âpmol/l and 7.18â±â1.93ânmol/l in the STEMI group (Pâ<â0.01 and Pâ=â0.33, respectively). Pearson correlation coefficient for OPG and Syntax Score, MGP and Syntax score was respectively 0.79 (Pâ<â0.01) and 0.18 (Pâ=â0.22) in the stable angina group, -0.03 (Pâ=â0.43) and 0.10 (Pâ=â0.5) in the STEMI group.Serum OPG and MGP levels were respectively 5.52â±â1.02âpmol/l and 7.56â±â1.42ânmol/l in diabetics, 4.3â±â0.8âpmol/l and 6.52â±â1.14ânmol/l in nondiabetics (Pâ<â0.05; Pâ<â0.05). CONCLUSION: OPG, in a relatively small group of patients with stable angina, correlates proportionally with the extent of coronary artery disease (CAD), as evaluated by the Syntax Score. Higher serum OPG levels can be observed in individuals with STEMI regardless of CAD burden. As for MGP, a potential role as marker of plaque calcification remains unproven.
Subject(s)
Angina, Stable/blood , Calcium-Binding Proteins/blood , Extracellular Matrix Proteins/blood , Myocardial Infarction/blood , Osteoprotegerin/blood , Aged , Angina, Stable/diagnostic imaging , Biomarkers/blood , Bone Density , Calcinosis , Coronary Angiography , Coronary Artery Disease/blood , Coronary Artery Disease/diagnostic imaging , Diabetic Angiopathies/blood , Humans , Male , Middle Aged , Myocardial Infarction/diagnostic imaging , Matrix Gla ProteinABSTRACT
While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Here we show that in Vk*MYC mice harboring oncogene-driven plasma cell proliferative disorder, disease appearance associated with substantial modifications of the BM microenvironment, including a progressive accumulation of both CD8+ and CD4+ T cells with a dominant T helper type 1 (Th1) response. Progression from asymptomatic to symptomatic MM was characterized by further BM accrual of T cells with reduced Th1 and persistently increased Th2 cytokine production, which associated with accumulation of CD206+Tie2+ macrophages, and increased pro-angiogenic cytokines and microvessel density (MVD). Notably, MVD was also increased at diagnosis in the BM of MGUS and SMM patients that subsequently progressed to MM when compared with MGUS and SMM that remained quiescent. These findings suggest a multistep pathogenic process in MM, in which the immune system may contribute to angiogenesis and disease progression. They also suggest initiating a large multicenter study to investigate MVD in asymptomatic patients as prognostic factor for the progression and outcome of this disease.