ABSTRACT
UNLABELLED: This observational study evaluated the occurrence of nonvertebral fragility fractures (NVFX) in over 4,000 men and women with osteoporosis treated with teriparatide (TPTD). The incidence of new NVFX decreased for patients receiving TPTD treatment for greater than 6 months. No new significant safety findings were observed in this large trial. INTRODUCTION: The Direct Assessment of Nonvertebral Fractures in Community Experience (DANCE) study evaluated the occurrence of NVFX in patients receiving TPTD for osteoporosis in a real-world setting. METHODS: DANCE is a multicenter, prospective, observational trial that examined the long-term effectiveness of TPTD in men and women with osteoporosis whom study physicians judged to be suitable for TPTD therapy. Patients received 20 µg TPTD per day by subcutaneous injection for up to 24 months and were followed for 24 months after treatment cessation. The incidence of patients experiencing a new NVFX, defined as a fracture associated with low trauma, was evaluated during four 6-month periods in both the treatment and cessation phases with >0 to ≤6 months serving as the reference. We also observed the spectrum and occurrence of serious adverse events. RESULTS: Of the 4,167 patients enrolled, 4,085 took one or more doses of TPTD (safety population); 3,720 were included in the efficacy analysis. The incidence of patients experiencing a NVFX was 1.42, 0.91, 0.70, and 0.81 % for the four treatment periods, respectively, and 0.80, 0.68, 0.33, and 0.33 % for the four periods after treatment cessation. Differences for each period were statistically significant compared with the reference period (first 6-month interval, each p < 0.05). No new significant safety findings were observed. CONCLUSIONS: In this study, the incidence of NVFX decreased for patients receiving TPTD for all three treatment periods >6 months compared to 0 to ≤6 months, and this trend persisted throughout the cessation phase. TPTD was generally well tolerated.
Subject(s)
Bone Density Conservation Agents/therapeutic use , Osteoporosis/drug therapy , Osteoporotic Fractures/prevention & control , Teriparatide/therapeutic use , Aged , Aged, 80 and over , Bone Density/drug effects , Bone Density Conservation Agents/administration & dosage , Bone Density Conservation Agents/adverse effects , Drug Administration Schedule , Female , Femur Neck/physiopathology , Hip Joint/physiopathology , Humans , Lumbar Vertebrae/physiopathology , Male , Middle Aged , Osteoporosis/epidemiology , Osteoporosis/physiopathology , Osteoporosis, Postmenopausal/drug therapy , Osteoporosis, Postmenopausal/epidemiology , Osteoporosis, Postmenopausal/physiopathology , Osteoporotic Fractures/epidemiology , Osteoporotic Fractures/physiopathology , Prospective Studies , Teriparatide/administration & dosage , Teriparatide/adverse effects , United StatesABSTRACT
UNLABELLED: The prospective, observational Direct Assessment of Nonvertebral Fracture in the Community Experience (DANCE) study shows that, among patients with risk factors for osteoporosis, women are more likely to be screened and to receive appropriate treatment than men. There needs to be greater awareness that osteoporosis affects both men and women. INTRODUCTION: The prospective, observational DANCE study evaluated teriparatide use in the mainland USA and Puerto Rico in patients with osteoporosis in a community setting. This analysis compares baseline characteristics of women and men that may contribute to differences in initiation of teriparatide therapy. METHODS: Investigators prescribed teriparatide 20 µg/day subcutaneous injection for ≤24 months to 3,698 patients (3,342 women, 356 men) whom they considered appropriate candidates for therapy. Study entry was guided by product labeling. Specific timing and frequency of office visits were not mandated. Treatment decisions were based on the clinical judgment of study investigators and local standards of care. RESULTS: At baseline, similar proportions of women and men had prior fragility fractures (57% and 59%, respectively) and comorbid conditions that increase fracture risk (83% and 84%, respectively). Women were older than men (mean age 68 vs. 65 year; P < 0.0001) and more likely to have received prior osteoporosis therapy (88% vs. 62%; P < 0.0001). Investigators prescribed teriparatide more often for women than men based on general frailty (21% vs. 16%; P = 0.0151), low body mass index (17% vs. 10%; P = 0.0005), and an inadequate response (58% vs. 36%; P < 0.0001) or intolerance to previous therapy (23% vs. 12%; P < 0.0001). Chronic glucocorticoid therapy was the reason investigators cited most frequently for initiating therapy more often in men than in women (17% vs. 10%; P < 0.0001) CONCLUSIONS: These results suggest that patients' gender may influence the reasons physicians initiate teriparatide therapy in a community setting.
Subject(s)
Bone Density Conservation Agents/therapeutic use , Osteoporosis/drug therapy , Osteoporotic Fractures/prevention & control , Teriparatide/therapeutic use , Age Distribution , Aged , Bone Density/drug effects , Drug Prescriptions/statistics & numerical data , Female , Femur Neck/physiopathology , Glucocorticoids/adverse effects , Hip Joint/physiopathology , Humans , Lumbar Vertebrae/physiopathology , Male , Middle Aged , Osteoporosis/diagnosis , Osteoporosis/physiopathology , Osteoporosis, Postmenopausal/diagnosis , Osteoporosis, Postmenopausal/drug therapy , Osteoporosis, Postmenopausal/physiopathology , Osteoporotic Fractures/physiopathology , Patient Selection , Prospective Studies , Secondary Prevention , Sex FactorsABSTRACT
The nuclear factor of activated T-cells (NFATp) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFATp. We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFATp. The phosphorylation state and nuclear/cytoplasmic location of NFATp were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFATp. These St-induced effects were inhibited by pretreatment with FK506, indicating that CaN activity was required for the observed effects on NFATp. Treatment of cells with ionomycin resulted in NFATp dephosphorylation and nuclear localization. Removal of ionomycin from the cells resulted in the reappearance of phosphorylated NFATp in the cytosol. St and BI also inhibited the re-accumulation of NFATp in the cytoplasm and its re-phosphorylation after ionomycin removal. The re-accumulation of NFATp in the cytosol after ionomycin withdrawal was shown to be energy- and temperature-dependent. Taken together, these results suggest that in unstimulated cells NFATp is actively maintained in the cytoplasm by kinases acting in opposition to basal CaN activity.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins , Phosphoprotein Phosphatases/metabolism , Protein Kinases/metabolism , Protein Processing, Post-Translational , T-Lymphocytes/metabolism , Transcription Factors/metabolism , Animals , Calcineurin , Cell Line , Cold Temperature , Enzyme Inhibitors/pharmacology , Immunosuppressive Agents/pharmacology , Indoles/pharmacology , Ionomycin/pharmacology , Ionophores/pharmacology , Kinetics , Lymphocyte Activation , Maleimides/pharmacology , Mice , NFATC Transcription Factors , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors , Staurosporine/pharmacology , T-Lymphocytes/drug effects , Tacrolimus/pharmacology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Cell Compartmentation , Cells, Cultured , Cytosol/metabolism , Humans , Immunologic Techniques , In Vitro Techniques , Ionomycin/pharmacology , Lymphocyte Activation/drug effects , Mice , Molecular Sequence Data , NFATC Transcription Factors , Nuclear Proteins/metabolism , Peptides/chemistry , Peptides/immunology , Phosphoproteins/metabolism , Phosphorylation , Phosphotyrosine , T-Lymphocytes/metabolism , Tacrolimus/pharmacology , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
Heat shock protein 56 (hsp56) was previously identified as an immunophilin based on its ability to specifically bind to FK506-Affi-Gel 10. In this report, we have quantitated human Jurkat T cell hsp56 binding to 3H-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that 3H-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-Sepharose column binds 3H-FK506 with an affinity of 19.4 +/- 4.6 nM, as compared to 23.2 +/- 6.8 nM for soluble hsp56. In competition experiments, the apparent affinity constant for rapamycin was 11.6 +/- 2.8 nM, using immobilized hsp56, and 17.3 +/- 7.7 nM, using the soluble hsp56 preparation. These results demonstrate that hsp56 binds FK506 and rapamycin with similar affinities, and suggest that hsp56 may play a role in mediating the cellular function of both of these drugs.
Subject(s)
Carrier Proteins/metabolism , Chromatography, Affinity/methods , DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Polyenes/metabolism , Tacrolimus/metabolism , Blotting, Western , Humans , Protein Binding , Radioligand Assay/methods , Sirolimus , Tacrolimus Binding Proteins , Tumor Cells, CulturedABSTRACT
Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.
Subject(s)
Carrier Proteins/metabolism , Heat-Shock Proteins/metabolism , Tacrolimus/metabolism , Animals , Antibodies/immunology , Antibody Specificity , Carrier Proteins/immunology , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Fluorescent Antibody Technique , Heat-Shock Proteins/immunology , Humans , Male , Rats , Rats, Sprague-Dawley , Tacrolimus Binding Proteins , Tissue DistributionABSTRACT
The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.
Subject(s)
Cell Nucleus/drug effects , Diglycerides/metabolism , Isoenzymes/metabolism , Protein Kinase C/metabolism , Thrombin/pharmacology , Animals , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cells, Cultured , Cricetinae , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Microscopy, Electron , Nuclear Proteins/metabolism , Phosphorylation , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
It was recently noted that the amino acid sequence of FK506 binding protein (FKBP-12) is nearly identical to that of an endogenous inhibitor of protein kinase C, PKCI-2. To follow up on this observation, we have tested the hypothesis that FKBP-12 is an inhibitor of PKC. The kinase activity of rat brain protein kinase C (PKC) was not inhibited by the presence of up to 700 micrograms recombinant human FKBP-12 per ml, in either the presence or absence of FK506. FKBP-12 also did not affect PMA-induced phosphorylation of an endogenous PKC substrate, an 80 kDa protein, in permeabilized cells. To test whether FKBP-12 could account for endogenous PKC inhibitory activity in cells, Jurkat cell lysate was chromatographed on an anion exchange column. A peak of PKC inhibitory activity was eluted at approximately 200 mM NaCl. As shown by both Western blots and FK506 binding activity, FKBP-12 was eluted only in the flow-through and wash fractions. These results demonstrate that FKBP-12 is clearly distinct from endogenous PKC inhibitory activity.
Subject(s)
Carrier Proteins/pharmacology , Protein Kinase C/antagonists & inhibitors , Tacrolimus/metabolism , Blotting, Western , Carrier Proteins/isolation & purification , Chromatography , Humans , Phosphorylation , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Tacrolimus Binding Proteins , Tumor Cells, CulturedABSTRACT
Extracts from human Jurkat cells or from calf thymus contain a 60-kDa protein that is bound to immobilized FK506. As expected, the NH2-terminal sequences of the 60-kDa protein from these two species were found to be nearly the same. We were surprised to discover, however, that the sequence of the human protein was identical to that of Hsp56, a heat shock protein of unknown function that has been shown to be a component of several steroid receptor complexes. Further analysis of the calf thymus protein revealed a peptide with homology to a region near the COOH terminus of both FKBP-12 and FKBP-13. It would appear, therefore, that this 60-kDa protein, or as we refer to it provisionally, "Hsp56," could have the capacity to bind FK506 directly. These observations lead us to speculate that "Hsp56" may mediate immunosuppression and inhibition of T-cell proliferation by FK506 and may do so via a cytosolic signal transduction pathway separate, but not necessarily exclusive, from that of FKBP-12 and FKBP-13.
Subject(s)
Carrier Proteins/metabolism , Heat-Shock Proteins/metabolism , Receptors, Steroid/metabolism , Tacrolimus/metabolism , Thymus Gland/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Cattle , Cell Line , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Heat-Shock Proteins/genetics , Heat-Shock Proteins/isolation & purification , Humans , Molecular Sequence Data , Molecular Weight , Protein Binding , Receptors, Steroid/genetics , Sequence Homology, Nucleic Acid , Tacrolimus Binding ProteinsABSTRACT
Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.
Subject(s)
Diglycerides/biosynthesis , Phosphatidylcholines/metabolism , Phosphatidylinositols/metabolism , Protein Kinase C/metabolism , Thrombin/pharmacology , Animals , Autoradiography , Blotting, Western , Cricetinae , Cricetulus , Digitonin/pharmacology , Enzyme Activation , Fibroblasts/drug effects , Phosphorylation , RatsABSTRACT
After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.
Subject(s)
Neurons/enzymology , Protein Kinase C/biosynthesis , Protein Kinases/biosynthesis , Cell Differentiation/drug effects , Enzyme Induction/drug effects , Humans , Neoplasm Proteins/biosynthesis , RNA, Messenger/biosynthesis , Teratoma/pathology , Tretinoin/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymologyABSTRACT
We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.
Subject(s)
Fibroblasts/enzymology , Nuclear Envelope/enzymology , Phorbol Esters/pharmacology , Protein Kinase C/metabolism , Animals , Antibodies, Monoclonal , Cell Fractionation , Cell Line , Cytoskeleton/enzymology , Cytoskeleton/ultrastructure , Fibroblasts/drug effects , Fibroblasts/ultrastructure , Fluorescent Antibody Technique , Isoenzymes/immunology , Isoenzymes/metabolism , Isoenzymes/physiology , Mice , Nuclear Envelope/drug effects , Nuclear Envelope/ultrastructure , Protein Kinase C/immunology , Protein Kinase C/physiology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Poly(A)+ RNA was isolated from Ascaris suum body wall muscle and translated in a cell-free rabbit reticulocyte lysate system. Specific antisera and immunoglobulins against the alpha-pyruvate dehydrogenase and dihydrolipoyl transacetylase components of ascarid pyruvate dehydrogenase complex were used to immunoprecipitate individual radiolabelled polypeptides from the in vitro translation mixtures. Both polypeptides appeared to be synthesized as preproteins about 1.5 and 8 kDa larger than the corresponding native proteins. Incubation of the dihydrolipoyl transacetylase preprotein with an ascarid high-speed mitochondrial supernatant fraction resulted in the formation of a polypeptide with apparent molecular weight intermediate in size between the preprotein and the native enzyme. This processing was insensitive to phenylmethylsulfonyl fluoride and leupeptin but was completely abolished by EDTA. These results suggest that in A.suum, as in other organisms, mitochondrial matrix proteins coded by the nuclear genome are synthesized as larger preproteins and processed by a specific, metal-dependent mitochondrial matrix protease.