ABSTRACT
High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short-term (24 h) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in 1-month-old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co-receptor Klotho, transcription factor mRNAs for early growth response-1 transcription factor (Egr-1), and c-fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p-FGFR1, p-FGFR3, KLOTHO, p-ERK1/2, C-FOS, and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. J. Cell. Physiol. 232: 610-616, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Antibodies, Neutralizing/therapeutic use , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factors/immunology , Hypophosphatemia/drug therapy , Hypophosphatemia/metabolism , Kidney/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction , Animals , Antibodies, Neutralizing/pharmacology , Bone Density , Cytochrome P-450 Enzyme System/metabolism , Early Growth Response Protein 1/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor-23 , Glucuronidase/metabolism , Hypophosphatemia/pathology , Klotho Proteins , Mice, Transgenic , Models, Biological , Molecular Weight , Phosphorylation , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co-delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co-delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly-l-Lysine (+) and poly l-Glutamic acid (-) (bCaP-PEM). Measurements of MC3T3-E1 proliferation and viability over time on bCaP-PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor [fibroblast growth factor-2 (FGF-2)] followed by a cytotoxic factor (antimycin A, AntiA). FGF-2 and AntiA both retained their bioactivity within bCaP-PEM, yet no release of FGF-2 or AntiA from bCaP-PEM was observed when cells were absent indicating a cell-mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off-target effects associated with diffusion-based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500-1509, 2017.
Subject(s)
Antimycin A , Biomimetic Materials , Calcium Phosphates , Coated Materials, Biocompatible , Drug Delivery Systems/methods , Fibroblast Growth Factor 2 , Polyelectrolytes , Animals , Antimycin A/chemistry , Antimycin A/pharmacokinetics , Antimycin A/pharmacology , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacokinetics , Biomimetic Materials/pharmacology , Calcium Phosphates/chemistry , Calcium Phosphates/pharmacokinetics , Calcium Phosphates/pharmacology , Cell Line , Cell Proliferation/drug effects , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacokinetics , Coated Materials, Biocompatible/pharmacology , Fibroblast Growth Factor 2/chemistry , Fibroblast Growth Factor 2/pharmacokinetics , Fibroblast Growth Factor 2/pharmacology , Mice , Polyelectrolytes/chemistry , Polyelectrolytes/pharmacokinetics , Polyelectrolytes/pharmacologyABSTRACT
Binge-eating behavior involves rapid consumption of highly palatable foods leading to increased weight gain. Feeding in binge disorders resembles other compulsive behaviors, many of which are responsive to N-acetylcysteine (NAC), which is a cysteine prodrug often used to promote non-vesicular glutamate release by a cystine-glutamate antiporter. To examine the potential for NAC to alter a form of compulsive eating, we examined the impact of NAC on binge eating in a rodent model. Specifically, we monitored consumption of standard chow and a high-fat, high carbohydrate western diet (WD) in a rodent limited-access binge paradigm. Before each session, rats received either a systemic or intraventricular injection of NAC. Both systemic and central administration of NAC resulted in significant reductions of binge eating the WD without decreasing standard chow consumption. The reduction in WD was not attributable to general malaise as NAC did not produce condition taste aversion. These results are consistent with the clinical evidence of NAC to reduce or reverse compulsive behaviors, such as, drug addiction, skin picking and hair pulling.
Subject(s)
Acetylcysteine/pharmacology , Binge-Eating Disorder/drug therapy , Disease Models, Animal , Feeding Behavior/drug effects , Feeding Behavior/psychology , Animals , Conditioning, Operant/drug effects , Diet, High-Fat , Injections, Intraventricular , Male , Rats , Rats, Sprague-DawleyABSTRACT
Since parathyroid hormone (PTH) increased FGF2 mRNA and protein expression in osteoblasts, and serum FGF-2 was increased in osteoporotic patients treated with PTH, we assessed whether the anabolic effect of PTH was impaired in Fgf2-/- mice. Eight-week-old Fgf2+/+ and Fgf2-/- male mice were treated with rhPTH 1-34 (80mug/kg) for 4 weeks. Micro-CT and histomorphometry demonstrated that PTH significantly increased parameters of bone formation in femurs from Fgf2+/+ mice but the changes were smaller and not significant in Fgf2-/- mice. IGF-1 was significantly reduced in serum from PTH-treated Fgf2-/- mice. DEXA analysis of femurs from Fgf2+/+, Fgf2+/-, and Fgf2-/- mice treated with rhPTH (160mug/kg) for 10 days showed that PTH significantly increased femoral BMD in Fgf2+/+ by 18%; by only 3% in Fgf2+/- mice and reduced by 3% in Fgf2-/- mice. We conclude that endogenous Fgf2 is important for maximum bone anabolic effect of PTH in mice.
Subject(s)
Bone and Bones/drug effects , Osteogenesis/drug effects , Parathyroid Hormone/pharmacology , Receptor, Fibroblast Growth Factor, Type 2/physiology , Animals , Bone Density/drug effects , Bone and Bones/metabolism , Female , Femur/ultrastructure , Humans , Male , Mice , Mice, Knockout , Receptor, Fibroblast Growth Factor, Type 2/deficiency , Receptor, Fibroblast Growth Factor, Type 2/genetics , Recombinant Proteins/pharmacology , Teriparatide/pharmacology , Tomography, X-Ray ComputedABSTRACT
We previously reported that deletion of the Fgf2 gene (Fgf2-/-) resulted in decreased bone mass in adult mice. This study examines the effect of haplo-insuffiency (Fgf2+/-) on bone loss in vertebrae from these mutant mice. Fgf2+/+ mice attained peak bone mass at 8-9 months of age. In contrast BMD was significantly reduced in vertebrae from adult (8-9) Fgf2+/- mice. Exogenous FGF-2 rescued reduced bone nodule formation in Fgf2+/- and Fgf2-/- cultures. Runx2 mRNA was reduced in cultures from Fgf2+/- and Fgf2-/- mice. FGF receptor2 mRNA and protein were markedly reduced in Fgf2+/- and Fgf2-/- mice. Decreased bone formation in Fgf2 mutant mice may correlate with impaired FGFR signaling, decreased Runx2 gene expression.
Subject(s)
Fibroblast Growth Factor 2/deficiency , Fibroblast Growth Factor 2/metabolism , Osteogenesis/physiology , Animals , Biomarkers , Body Weight , Bone Density , Cell Differentiation , Core Binding Factor Alpha 1 Subunit/genetics , Fibroblast Growth Factor 2/genetics , Haplotypes , Mice , Mice, Knockout , Osteoclasts/cytology , Osteoclasts/metabolism , RNA, Messenger/genetics , Tissue Culture TechniquesABSTRACT
The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF-2 at the time (1993), only a few reported a role of FGF-2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF-2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF-2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF-2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation.
Subject(s)
Craniosynostoses/metabolism , Fibroblast Growth Factors/metabolism , Osteochondrodysplasias/metabolism , Signal Transduction , Animals , Apoptosis , Bone Development , Cell Differentiation , Disease Models, Animal , Humans , Ligands , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , Models, Genetic , Mutation , Osteoblasts/metabolism , Protein IsoformsABSTRACT
Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.
Subject(s)
Bone Diseases, Metabolic/physiopathology , Calcification, Physiologic , Fibroblast Growth Factor 2/biosynthesis , Gene Expression , Osteoblasts/metabolism , Animals , Bone Diseases, Metabolic/genetics , Bone Diseases, Metabolic/pathology , Bone Remodeling/genetics , Calcification, Physiologic/genetics , Fibroblast Growth Factor 2/genetics , Humans , Mice , Mice, Transgenic , Osteoblasts/cytologyABSTRACT
Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.
Subject(s)
Fibroblast Growth Factor 2/biosynthesis , Osteoblasts/metabolism , Protein Isoforms/biosynthesis , Animals , Blotting, Northern , Blotting, Western , Cell Division/genetics , Cell Survival , Cloning, Molecular , Fibroblast Growth Factor 2/genetics , Fluorescent Antibody Technique , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins , Humans , Luminescent Proteins , Microscopy, Fluorescence , Protein Isoforms/genetics , RNA, Messenger/analysis , Rats , Time Factors , Transfection , TransgenesABSTRACT
Fibroblast growth factor 2 (FGF2) and noggin are two unrelated ligands of two distinctly different signaling pathways that have a similar inhibitory effect on osteoblast differentiation. Because of their differences, we postulated that they probably acted at a different stage within the osteoprogenitor differentiation pathway. This study was performed on primary murine bone cell cultures under conditions where alkaline phosphatase (AP) and type I collagen expression (Col1a1) were observed by day 7 (preosteoblast stage), followed by bone syaloprotein (BSP) at day 11 (early osteoblast) and osteocalcin (OC) by day 15-18 (mature osteoblast stage). FGF2 completely inhibited expression of AP and the mRNA transcript for Col1a1, while noggin showed only a partial inhibition of these markers of preosteoblast differentiation. However, the markers of differentiated osteoblasts (BSP and OC) were completely inhibited in both the FGF2 and noggin treated cultures, suggesting that noggin acts at later point in the osteoprogenitor differentiation pathway than FGF2. To further verify that the inhibition was occurring at a different stage of osteoblasts development, primary cultures derived from transgenic mice harboring segments of the collagen promoter driving green fluorescent protein (GFP) that activate at different levels of osteoblast differentiation were analyzed. Consistent with the endogenous markers, pOBCol3.6GFP and pOBCOL2.3GFP transgene activity was completely inhibited by continuous addition of FGF2, while noggin showed partial inhibition of pOBCol3.6GFP and complete inhibition of the pOBCol2.3GFP transgene. Upon removal of either agent, endogenous and GFP markers of osteoblast differentiation reappeared although at a different temporal pattern. This work demonstrates that FGF2 and noggin can reversibly modulate osteoblast lineage differentiation at different maturational stages. These agents may be useful to enrich for and maintain a population of osteoprogenitor cells at a defined stage of differentiation.
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Growth Inhibitors/pharmacology , Osteoblasts/physiology , Stem Cells/physiology , Animals , Carrier Proteins , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Fibroblast Growth Factor 2/genetics , Genetic Therapy , Green Fluorescent Proteins , Luminescent Proteins , Mice , Mice, Transgenic , Osteoblasts/drug effects , Proteins/antagonists & inhibitors , Proteins/genetics , Proteins/pharmacology , Stem Cells/drug effects , Transgenes/drug effectsABSTRACT
We evaluated, by confocal laser scanning microscopy, the actin cytoskeleton of immortalized rat Py1a osteoblasts treated with phthalate esters (butyl benzyl phthalate, BBP and dibutyl phthalate, DBP), endocrine disruptors with estrogenic activity. We observed some peculiar modifications of actin cytoskeleton and cells changing from a spindle shape to a rounded form. In particular, F-actin formed thick bundles around the cell membrane but only a weak labeling was observed in rounded cells. Also influence on apoptosis and short-term effects on FGF-2 were studied. It was found that BBP and DBP exert their action in a similar way, act in a transient manner and do not induce apoptosis.
Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Dibutyl Phthalate/pharmacology , Osteoblasts/metabolism , Phthalic Acids/pharmacology , Teratogens/pharmacology , Animals , Cell Line , DNA Fragmentation , Fibroblast Growth Factor 2/metabolism , Fluorescein , Microscopy, Confocal , Osteoblasts/drug effects , RatsABSTRACT
Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-beta; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-beta. TGF-beta increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 microM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-beta did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of approximately 18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-beta. Increased FGF-2 mRNA and protein expression in response to TGF-beta was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-beta showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-beta treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-beta regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-beta modulates the cellular localization of FGF-2 and its receptors.
Subject(s)
Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Osteoblasts/drug effects , Osteoblasts/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Transforming Growth Factor beta/pharmacology , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Microscopy, Immunoelectron , Osteoblasts/ultrastructure , Protein Kinase C/antagonists & inhibitors , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptor, Fibroblast Growth Factor, Type 2ABSTRACT
FGF-2 stimulates bone formation in vitro and in vivo in rats. However, there are limited studies in mice and no data on the mechanism(s) by which FGF-2 induces bone formation. We assessed whether short-term FGF-2 treatment of marrow stromal cells from young mice would increase alkaline phosphatase-positive (ALP), mineralized colony formation and expression of genes important in osteoblast maturation. Short-term treatment with FGF-2 (0.01-1.0 nM) for the first 3 days of a 14- or 21-day culture period increased the number of ALP mineralized colonies in bone marrow stromal cells. FGF-2 (0.1 nM) increased the mRNAs for type 1 collagen: osteocalcin, runt domain/core binding factor, PTH/PTHR receptor, and insulin-like growth factor 1 (IGF-1) at 14 and 21 days. We conclude that short-term FGF-2 treatment enhances osteoblast maturation in vitro. Furthermore, the anabolic effect of FGF-2 may be attributed in part to regulation of IGF-1 in osteoblasts.
Subject(s)
Bone Marrow Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factor 2/physiology , Insulin-Like Growth Factor I/biosynthesis , Neoplasm Proteins , RNA, Messenger/metabolism , Stromal Cells/metabolism , Animals , Bone and Bones/metabolism , Cell Division , Cells, Cultured , Collagen Type I/biosynthesis , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Fibroblast Growth Factor 2/pharmacology , Mice , Mice, Inbred C57BL , Osteocalcin/biosynthesis , Parathyroid Hormone/biosynthesis , Rats , Time Factors , Transcription Factors/metabolismABSTRACT
OBJECTIVE: The major aim of this study was to determine whether a combination of hand preference inventories and hand performance measures identifies distinct handedness groups. If distinct groups are identified, then these subgroupings can be used in future studies to learn more about the neurobiology of these distinct handedness groups. BACKGROUND: Although most individuals classify themselves as right- or left-handed, it is not entirely clear whether handedness should be determined based on preference inventories, hand performance tasks, or a combination of these measures. Given that hand preference is linked in part to hemispheric specialization of language, it is important to clearly define hand preference groups if lateralized differences between right- and left-handers are to be explored. Healthy adult right- and left-handers were examined from a multivariate perspective in an attempt to determine whether handedness subgroups exist within performance data. METHOD: Hand preference of 62 right- and left-handed male and female adults was assessed using items from Briggs and Nebes' and Oldfield's handedness inventories. Individuals were assigned to right- and left-hand preference groups, both by visually inspecting the distribution of preference scores and via cluster analysis. Asymmetries in performance of unimanual motor tasks (grooved pegboard, finger-tapping, and grip strength) were then examined using a multivariate approach. RESULTS: Sixteen items from the two-handedness inventories were used to determine preference-based handedness groups. Two non-overlapping groups, right- and left-hand preference, were identified. Writing hand was highly correlated with hand-preference group, as only three individuals in the entire sample wrote with the non-preferred hand. The expected unimodal distributions of performance asymmetry scores, known as laterality quotients (LQs), were seen. However, when those LQs were viewed from a multivariate perspective, distinct performance-based groups emerged. In more than 90% of the observed cases, the performance-based groups corresponded to preference-based groups. No sex differences were found; the relationship between preference and performance measures was not significantly different for men and women. CONCLUSIONS: Writing hand was highly correlated with scores from a hand preference inventory. In contrast, the use of a single hand performance measure, finger tapping or pegboard, did not always correctly classify an individual as right- or left-handed. However, when both of these hand performance measures were used together. individuals were correctly classified as right- or left-handed. Using this approach, two approximately non-overlapping groups, right- and left-handers, emerged. Thus, handedness is probably not a one-dimensional trait or behavior, and must be defined using multiple measures that assess different aspects of hand preference and performance. The implications for hemispheric specialization of language and neural asymmetry research are discussed.
Subject(s)
Functional Laterality/physiology , Adult , Choice Behavior , Female , Humans , Male , Middle Aged , Neuropsychological Tests , Psychomotor Performance/physiology , Random AllocationABSTRACT
Interleukin-1 (IL-1) and basic fibroblast growth factor (FGF-2) are potent stimulators of osteoclast formation. However, the role of FGF-2 in the responses to IL-1 in bone has not been reported. We examined the effect of IL-1 on FGF-2 mRNA and protein expression in human osteosarcoma MG-63 osteoblasts, normal human osteoblasts (NHOB), and osteoblasts from osteoarthritic patients (F2 and F13). IL-1 increased FGF-2 mRNA expression in osteoblasts within 1.5 to 3 h. Multiple FGF-2 protein isoforms were expressed in human osteoblasts. Twenty-four hours of treatment of MG-63 and NHOB cells with IL-1 increased the high-molecular-weight(HMW, 22/24 kDa) and low-molecular-weight (LMW, 18 kDa) FGF-2 proteins intracellularly. In contrast, IL-1 preferentially increased the LMW protein signal intracellularly as well as on the cell surface of F2 and F13 osteoblasts. We conclude that IL-1 is a major stimulator of FGF-2 expression in human osteoblasts. Furthermore, selective increases in the exportable LMW protein in osteoblasts from osteoarthritic patients may be of clinical relevance.
Subject(s)
Fibroblast Growth Factor 2/genetics , Interleukin-1/pharmacology , Osteoblasts/drug effects , RNA, Messenger/genetics , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation/drug effects , Humans , Osteoarthritis/genetics , Osteoblasts/metabolism , Protein Isoforms/metabolism , Tumor Cells, CulturedABSTRACT
Exposure of the Py1a rat osteoblastic cells to butyl benzyl phthalate (BBP) and dibutyl phthalate (DBP) showed that these endocrine disrupting chemicals (EDC) strongly and reversibly affect the cytoplasmic fibroblast growth factor-2 (FGF-2) translocation into the nucleus in a dose-dependent and time-related manner. Stimulation of cells with high concentrations of BBP or DBP for short timing gave results comparable to those of cells treated with low concentrations for long timing. By confocal laser scanning microscope (CLSM) analysis it was found that the first relevant effect resulted in an accumulation of FGF-2 near the nuclear envelope, sometimes in the shape of clusters; the growth factor was then translocated into the nucleus and, finally, after long periods of exposure, the basal nuclear and cytoplasmic binding, typical of unstimulated cells, was re-established. In addition it was found that phthalate esters did not affect the FGF receptor 2 (FGFR-2) but decreased Con A binding indicating a possible inhibition of collagen fiber assembly. The different concentrations and timing of exposure of BBP and DBP affected the FGF-2 modulation in a similar way. Noticeable cumulative effects of BBP and DBP were not observed.
Subject(s)
Esters/pharmacology , Fibroblast Growth Factor 2/metabolism , Osteoblasts/metabolism , Phthalic Acids/pharmacology , Animals , Cell Line , Cell Nucleus/metabolism , Collagenases/metabolism , Concanavalin A/pharmacology , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Immunohistochemistry , Lectins/pharmacology , Microscopy, Confocal , Microscopy, Fluorescence , Protein Binding , Protein Transport , Rats , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Fibroblast Growth Factor/metabolism , Time FactorsABSTRACT
Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.
Subject(s)
Bone and Bones/physiology , Fibroblast Growth Factor 2/physiology , Alkaline Phosphatase/metabolism , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Femur , Fibroblast Growth Factor 2/genetics , Mice , Mice, Knockout , Osteoblasts/cytology , Osteogenesis/physiology , RNA, Messenger , Skull/cytology , TibiaABSTRACT
We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1-34) (10-8M) or forskolin (FSK; 10-5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.
Subject(s)
Fibroblast Growth Factor 2/genetics , Gene Expression Regulation, Developmental , Osteoblasts/metabolism , Parathyroid Hormone/physiology , Protein-Tyrosine Kinases , RNA, Messenger/biosynthesis , Receptors, Fibroblast Growth Factor/genetics , Animals , Cells, Cultured , Cycloheximide/pharmacology , Fluorescent Antibody Technique , Humans , Mice , Protein Synthesis Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 2 , Receptor, Fibroblast Growth Factor, Type 3 , Receptor, Fibroblast Growth Factor, Type 4 , Transcription, Genetic , TransfectionSubject(s)
Fluorescein-5-isothiocyanate/chemistry , Osteoblasts/cytology , Osteoblasts/metabolism , Peanut Agglutinin/metabolism , Animals , Biomarkers/analysis , Cell Cycle , Cell Line , Cell Nucleus/metabolism , Fluorescent Dyes/metabolism , Mitosis , Peanut Agglutinin/chemistry , Rats , Rhodamines/chemistryABSTRACT
We examined the effect of PGs, particularly PGF2alpha, on basic fibroblast growth factor-2 (FGF-2) messenger RNA (mRNA) and protein in the rat osteoblastic cell line Py1a and in fetal rat calvariae. Py1a cells expressed multiple FGF-2 mRNA transcripts. PGF2alpha dose-dependently increased the 6-kb transcript at 6 h. The selective PGF2alpha agonist, fluprostenol (Flup), was more potent than PGF2alpha. Phorbol myristate acetate (10(-6) M) also increased a 6-kb mRNA at 6 h. By immunofluorescence microscopy, Flup increased perinuclear staining for FGF-2 protein at 6 h and nuclear labeling at 24 h. Immunogold labeling of calvariae revealed that treatment with Flup for 3 h caused a transition of FGF expression from matrix to cells and an increase in cytoplasmic labeling for FGF-2 protein in periosteal cells and in osteoblasts. After treatment with Flup for 24 h, nuclear labeling was marked in periosteal cells and in osteoblasts, and a further increase in cytoplasmic labeling for FGF-2 was noted in osteocytes, periosteal cells, and osteoblasts. We conclude that PGs can increase FGF-2 mRNA and protein in bone cells. Because the effect of Flup was mimicked by phorbol myristate acetate, we hypothesize that PGs' regulation of FGF-2 is mediated by a PGF2alpha-selective receptor acting through protein kinase C. Hence, effects of PGs on bone remodeling may be mediated, in part, by endogenous FGF-2.
Subject(s)
Bone and Bones/metabolism , Fibroblast Growth Factor 2/biosynthesis , Prostaglandins/physiology , Animals , Bone and Bones/drug effects , Bone and Bones/ultrastructure , Cells, Cultured , Dinoprost/pharmacology , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation/drug effects , Luteolytic Agents/pharmacology , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/ultrastructure , Prostaglandins F, Synthetic/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
We determined the effect of basic fibroblast growth factor (bFGF) on osteoclast-like cell (OCL) formation in bone marrow cultures using C57BL/6 mice. Cells were cultured for 7 days with or without bFGF at various concentrations or 10(-8) mol/L 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. bFGF dose-dependently increased OCL formation per well (10(-10) mol/ L = 40 +/- 2; 10(-9) mol/L = 146 +/- 13; 10(-8) mol/L = 156 +/- 12) compared with control (< 7 per well). The effects of bFGF at 10(-9) and 10(-8) mol/L were similar to that of 10(-8) mol/L 1,25(OH)2D3 (154 +/- 11 per well). OCLs formed by bFGF were multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive, expressed calcitonin receptors, and formed characteristic resorption pits. We also determined whether bFGF enhanced OCL formation during the early proliferative or late differentiating phases of the cultures. When bFGF (10(-8) mol/L) was added only on days 1-2 or days 3-4 of 6 day cultures, there was a significant increase in OCL formation. In contrast, when bFGF was added only on days 5-6 few OCLs formed. Addition of bFGF at days 1-6 or days 1-2 and days 5-6 caused similar increases in OCL formation, which were greater than OCL formation induced by treatment for days 1-2 or days 1-4. We examined the production of prostaglandin E2 (PGE2) in the cultures because bFGF is a potent stimulator of PGE2 synthesis in bone, and PGE2 stimulates OCL formation. bFGF treatment significantly increased PGE2 levels in 7 day cultures (controls = 1.4 +/- 0.1 nmol/L, 10(-8) mol/L bFGF = 132.5 +/- 0.7 nmol/L). In addition, treatment of marrow cultures with the prostaglandin synthesis inhibitors, indomethacin or NS-398 (both at 10(-6) mol/L), completely blocked bFGF-induced OCL formation. We conclude that bFGF stimulates OCL formation in C57BL/6 bone marrow cultures by mechanisms that require prostaglandin synthesis. This pathway is likely to be one mechanism by which bFGF stimulates resorption.