Richard J. Murrills

The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: Validation of a simplified light microscope assay of bone resorption☆

The aim of this study was to investigate whether a cyclic AMP-mediated inhibitory mechanism is present in embryonic chick osteoclasts and to extend data implicating cyclic AMP in the inhibition of neonatal rat osteoclasts. Dibutyryl cyclic AMP ((Bu)2cAMP) (5 x 10(-4) M and above) and isobutylmethylxanthine (IBMX) (10(-4) M and above) reduced the number of pits made in slices of devitalized bovine cortical bone by chick osteoclasts over 24 h. The effect of forskolin (FSK) on chick osteoclasts was biphasic, 10(-5) M producing a weak and variable reduction in pit number while 10(-6) M and 10(-7) M stimulated resorption. Doses of FSK (10(-5) M) and (Bu)2cAMP (3 x 10(-4) M), which individually produced no consistent significant effect, produced a synergistic and highly significant reduction in pit number when used in combination, implying that these agents were acting through a common mechanism, presumably cyclic AMP. Stimulatory doses of FSK were associated with increased osteoclast numbers, implicating cyclic AMP in the formation of osteoclasts. In comparative experiments using neonatal rat osteoclasts, (Bu)2cAMP (10(-4) M and above), IBMX (10(-3) M) and FSK (10(-7) M and above) all reduced the number of pits excavated. Strongly inhibitory doses of these agents caused contraction of chick osteoclasts into a hemispherical shape; contraction of rat osteoclasts into a stellate shape occurred with (Bu)2cAMP and FSK, but not with IBMX. Our results implicate cyclic AMP in the inhibition of both rat and chick osteoclasts, and show that pit counting in the light microscope is a valid method of analyzing the disaggregated osteoclast resorption assay.

(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: A Wingless β-Catenin Agonist That Increases Bone Formation Rate

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.

Identification of a PTH regulated gene selectively induced in vivo during PTH-mediated bone formation.

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 PGE2 and 1,25 dihydroxy vitamin D3. 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. J. Cell. Biochem. 98: 1203–1220, 2006.

Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

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.

Isolation and culture of osteoclasts and osteoclast resorption assays

The central role that bone resorption plays in the pathogenesis of human disorders such as osteoporosis, rheumatoid arthritis, Paget’s disease, hypercalcaemia of malignancy and osteopetrosis has long prompted the scientific community to inquire into the biology of the osteoclast. Over time, the investigative tools have changed. In recent years, the advances seen first in in vitro organ culture of bone, then in the isolation, purification and culture of selected cell types and ultimately in the molecular biology of osteoclasts have led to a rapidly improving understanding of the molecular engines comprising the osteoclast and its interactions with neighbouring and distant cell types. This chapter will begin by outlining the techniques that have been developed for isolating and purifying osteoclasts before concentrating on the technique with which we have most of our personal experience, the assay of bone resorption using isolated osteoclasts on bone slices, and finally closing with a summary of the advances that have been possible using these techniques. Our definition of an osteoclast is based upon function, namely that the ability to resorb a pit under conducive conditions is an essential requirement.

LACK OF SIGNIFICANT EFFECT OF CARBOXYL-TERMINAL PARATHYROID HORMONE-RELATED PEPTIDE FRAGMENTS ON ISOLATED RAT AND CHICK OSTEOCLASTS

Parathyroid hormone-related protein (PTHrP) fragments 107–111, 107–138, and 107–139 were all found to be without consistent significant inhibitory effect on the resorptive activity of isolated rat and chick osteoclasts over the dose range 10-13 M-10-9 M. In the rat, these results contrasted with a strong and significant inhibition (100%) by calcitonin. Our results differ from that previously reported for C-terminal fragments of PTHrP by Fenton et al. [1–3], who noted up to a 70% inhibition of resorptive activity of isolated rat or chick osteoclasts at femtomolar doses and greater. It is possible that the lack of response observed in our osteoclast assays is due to unknown variables in the bone slice assay that influence the responsiveness of isolated osteoclasts to these fragments.

A cell‐based Dkk1 binding assay reveals roles for extracellular domains of LRP5 in Dkk1 interaction and highlights differences between wild‐type and the high bone mass mutant LRP5(G171V)

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 125I‐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 β‐propellers plus the ligand binding domain, or the first β‐propeller, each inhibited Dkk1 binding, with mean IC50s of 10 and 196u2009nM, respectively. The extracellular domain of Kremen‐2 (“soluble Kremen”) was a weaker antagonist (mean IC50 806u2009nM). 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 β‐propellers plus the ligand binding domain, or the first β‐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. J. Cell. Biochem. 108: 1066–1075, 2009.

Hit to lead studies on (hetero)arylpyrimidines—Agonists of the canonical Wnt-β-catenin cellular messaging system

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.

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: Passage dependent effects on G protein interactions

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS‐2 cells varies as a function of cell passage. In early passage (EP) cells (<u20096), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (>u200922), 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 [32P]ADP‐ribosylation of its substrates, GTPase activity, as well as FOR‐dependent ACA, were equivalent in EP and LP. The relative amounts of Gαi and Gαs proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [32P]ADP‐ribosylation, were quantitatively similar in EP and LP. Western blot levels of Gαs and Gαi proteins were not influenced by prior exposure to PT. Both PT and CT dependent [32P]ADP‐ribosylation were dose‐dependently decreased following exposure to PT. However, the PT‐dependent decline in CT‐dependent [32P]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. Gαs 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. J. Cell. Physiol. 193: 10–18, 2002.

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

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 µM) 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. J. Cell. Biochem. 106: 887–895, 2009.