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1.
J Pharmacol Exp Ther ; 340(3): 676-87, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22171089

ABSTRACT

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.


Subject(s)
Osteogenesis/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , src-Family Kinases/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Differentiation , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects
2.
Proc Natl Acad Sci U S A ; 107(18): 8457-62, 2010 May 04.
Article in English | MEDLINE | ID: mdl-20404172

ABSTRACT

Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A(-/-) knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease-related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N-methyl-D-aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate alpha-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor-associated proteins stargazin (gamma2) and gamma8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.


Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Hippocampus/enzymology , Mental Disorders/enzymology , 3',5'-Cyclic-GMP Phosphodiesterases/deficiency , 3',5'-Cyclic-GMP Phosphodiesterases/genetics , Animals , Behavior, Animal , Female , Gene Expression Regulation, Enzymologic , Glutamine/metabolism , Hippocampus/pathology , Male , Mental Disorders/genetics , Mental Disorders/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , RNA, Messenger/genetics , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction , Social Behavior
3.
Bioorg Med Chem Lett ; 20(1): 366-70, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19897365

ABSTRACT

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.


Subject(s)
Imidazoles/chemistry , Pyrimidines/chemistry , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Bone Development/drug effects , Cell Line, Tumor , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Skull/metabolism , Wnt Proteins/agonists , Wnt Proteins/genetics , Wnt3 Protein , Wnt3A Protein , beta Catenin/agonists
4.
J Med Chem ; 52(22): 6962-5, 2009 Nov 26.
Article in English | MEDLINE | ID: mdl-19856966

ABSTRACT

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.


Subject(s)
Osteogenesis/drug effects , Piperidines/pharmacology , Pyrimidines/pharmacology , Wnt Proteins/agonists , beta Catenin/agonists , Animals , Catalytic Domain , Cell Line, Tumor , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/chemistry , Glycogen Synthase Kinase 3 beta , Humans , Mice , Models, Molecular , Piperidines/administration & dosage , Piperidines/pharmacokinetics , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Rats , Signal Transduction/drug effects , Wnt Proteins/metabolism , beta Catenin/metabolism
5.
J Cell Biochem ; 108(5): 1066-75, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19746449

ABSTRACT

Dkk1 is a secreted antagonist of the LRP5-mediated Wnt signaling pathway that plays a pivotal role in bone biology. Because there are no well-documented LRP5-based assays of Dkk1 binding, we developed a cell-based assay of Dkk1/LRP5 binding using radioactive (125)I-Dkk1. In contrast to LRP6, transfection of LRP5 alone into 293A cells resulted in a low level of specific binding that was unsuitable for routine assay. However, co-transfection of LRP5 with the chaperone protein MesD (which itself does not bind Dkk1) or Kremen-2 (a known Dkk1 receptor), or both, resulted in a marked enhancement of specific binding that was sufficient for evaluation of Dkk1 antagonists. LRP5 fragments comprising the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, each inhibited Dkk1 binding, with mean IC(50)s of 10 and 196 nM, respectively. The extracellular domain of Kremen-2 ("soluble Kremen") was a weaker antagonist (mean IC(50) 806 nM). We also found that cells transfected with a high bone mass mutation LRP5(G171V) had a subtly reduced level of Dkk1 binding, compared to wild type LRP5-transfected cells, and no enhancement of binding by MesD. We conclude that (1) LRP5-transfected cells do not offer a suitable cell-based Dkk1 binding assay, unless co-transfected with either MesD, Kremen-2, or both; (2) soluble fragments of LRP5 containing either the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, antagonize Dkk1 binding; and (3) a high bone mass mutant LRP5(G171V), has subtly reduced Dkk1 binding, and, in contrast to LRP5, no enhancement of binding with MesD.


Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , LDL-Receptor Related Proteins/metabolism , Molecular Chaperones/metabolism , Receptors, Cell Surface/metabolism , Receptors, LDL/metabolism , Amino Acid Substitution , Binding Sites , Biological Assay , Bone and Bones/metabolism , Cell Line , Humans , LDL-Receptor Related Proteins/chemistry , LDL-Receptor Related Proteins/genetics , Low Density Lipoprotein Receptor-Related Protein-5 , Low Density Lipoprotein Receptor-Related Protein-6 , Molecular Chaperones/genetics , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Cell Surface/genetics
6.
J Cell Biochem ; 106(5): 887-95, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19180574

ABSTRACT

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE-Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos-2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH-induced activation of the CRE. Activators of non-cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE-luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H-89 (10 microM) almost completely blocked PTH-induced activation of the CRE-reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time-dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH-induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN-93 had no significant effect on the response to PTH. We conclude that non-cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH.


Subject(s)
Adenosine Monophosphate/metabolism , Cyclic AMP/metabolism , Parathyroid Hormone/pharmacology , Response Elements/drug effects , Calcium/metabolism , Cell Line, Tumor , Epidermal Growth Factor/metabolism , Humans , Protein Kinase C/metabolism , Signal Transduction
7.
J Cell Biochem ; 98(5): 1203-20, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16514668

ABSTRACT

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well-established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH-regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100-fold (maximal) by a PTH 1-34 anabolic treatment regimen in a time-dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR-106 osteoblastic cells show that PTH 1-34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1-34, BAALC expression can also be induced by other bone forming factors including PGE(2) and 1,25 dihydroxy vitamin D(3). We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3-E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1-34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function.


Subject(s)
Gene Expression Regulation/drug effects , Osteogenesis/drug effects , Osteogenesis/genetics , Parathyroid Hormone/pharmacology , Amino Acid Sequence , Animals , Biomarkers , Calcitriol/pharmacology , Cell Differentiation , Cells, Cultured , Conserved Sequence , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Dinoprostone/pharmacology , Female , Humans , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Rats , Rats, Sprague-Dawley , Sequence Alignment , Signal Transduction
8.
J Biomol Screen ; 10(4): 304-13, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15964931

ABSTRACT

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF-generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.


Subject(s)
Gene Expression Regulation , Protein Engineering , Transcription Factors/physiology , Zinc Fingers , Amino Acid Sequence , Base Sequence , Cell Line , DNA Primers , Humans , Molecular Sequence Data , RNA, Messenger/genetics , Receptor, Parathyroid Hormone, Type 1/chemistry , Receptor, Parathyroid Hormone, Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/chemistry
9.
Bone ; 35(6): 1263-72, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15589208

ABSTRACT

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1-29) and a modified PTH(1-21) (MPTH(1-21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1-29), although active, was approximately 20-fold less potent than PTH(1-34) in a rat model of osteogenesis while MPTH(1-21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.


Subject(s)
Calcium Signaling/physiology , Cyclic AMP/physiology , Parathyroid Hormone/physiology , Peptide Fragments/physiology , Animals , Bone Density/drug effects , Bone Density/physiology , Calcium Signaling/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Parathyroid Hormone/metabolism , Parathyroid Hormone/pharmacology , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Receptors, Parathyroid Hormone/metabolism , Receptors, Parathyroid Hormone/physiology
10.
J Cell Physiol ; 193(1): 10-8, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12209875

ABSTRACT

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.


Subject(s)
Adenylyl Cyclases/metabolism , Bone Neoplasms/metabolism , GTP-Binding Proteins/metabolism , Osteoblasts/metabolism , Osteosarcoma/metabolism , Parathyroid Hormone/pharmacology , Adenylate Cyclase Toxin , Cell Differentiation/drug effects , Cholera Toxin/pharmacology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , GTP Phosphohydrolases/metabolism , Guanylyl Imidodiphosphate/pharmacology , Humans , Osteoblasts/drug effects , Pertussis Toxin , Protein Synthesis Inhibitors/pharmacology , Time Factors , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology
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