Rachelle J. Sells Galvin

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled‐1 (FZD‐1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low‐density lipoprotein‐receptor‐related protein 5 (LRP5), LRP6, FZD‐1, Dickkopf‐1 (Dkk‐1), and Kremen‐1 (KRM‐1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1–38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD‐1 and decreased LRP5 and Dkk‐1. These effects in UMR 106 cells were associated with an increase in β‐catenin as measured by Western blots and resulted in functional activation (three to six‐fold) of a downstream Wnt responsive TBE6‐luciferase (TCF/LEF‐binding element) reporter gene. Activation of the TBE6‐luciferase reporter gene by PTH (1–38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1–31), PTH‐related protein (1–34), and forskolin, but both PTH (3–34) and (7–34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP‐PKA pathway through the differential regulation of the receptor complex proteins (FZD‐1/LRP5 or LRP6) and the antagonist (Dkk‐1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE2)), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the β-galactosidase reporter gene (pOPG5.9βgal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9βgal reporter construct led to 39-, 7-, and 16-fold increases in β-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Roles of stromal cell RANKL, OPG, and M‐CSF expression in biphasic TGF‐β regulation of osteoclast differentiation

To better understand the complex roles of transforming growth factor‐beta (TGF‐β) in bone metabolism, we examined the impact of a range of TGF‐β concentrations on osteoclast differentiation. In co‐cultures of support cells and spleen or marrow osteoclast precursors, low TGF‐β concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF‐β on macrophage colony stimulating factor (M‐CSF), receptor activator of NF‐κB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M‐CSF expression. RANKL expression was elevated at low TGF‐β concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF‐β dose. Thus, low TGF‐β concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF‐β concentrations, there is sufficient M‐CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF‐β concentrations, the RANKL/OPG ratio and M‐CSF expression were both repressed and there was no differentiation. We examined whether TGF‐β‐mediated repression of osteoclasts differentiation is due to these changes by adding M‐CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF‐β impacts on osteoclast precursors by culturing spleen or marrow cells with M‐CSF and RANKL. TGF‐β treatment dose‐dependently stimulated osteoclast differentiation. These data indicate that low TGF‐β levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF‐β levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M‐CSF expression regulation.

Proliferating cells in the primary spongiosa express osteoblastic phenotype in vitro

We have shown that intermittent parathyroid hormone (PTH) treatment targets proliferating cells in the primary spongiosa of trabecular bone of young rats, resulting in an increased number of osteoblasts. To further characterize these proliferating osteoprogenitor cells, bromodeoxyuridine (BrdUrd) incorporated in vivo, was used as a marker to identify and isolate cells for in vitro studies. Proliferating cells were labeled in vivo in young rats with BrdUrd and 24 h later were isolated by trypsinization of sections of the primary spongiosa of the distal femur metaphysis. Within 12 h of isolation, BrdUrd+ cells formed distinct foci containing 20-500 cells with fibroblast morphology. Stimulation of proliferation as determined by [3H]-thymidine incorporation was observed for these cells in response to fetal bovine serum, platelet derived growth factor, and transforming growth factor beta-1. Neither insulin-like growth factor-1 (IGF-1) nor insulin stimulated proliferation PTH (1-34) and dexamethasone inhibited proliferation. The effects of PTH and dexamethasone were additive. Cells expressed the osteoblast phenotype as evidenced by synthesis of type I collagen, expression of high alkaline phosphatase activity, and production of increased intracellular cAMP in response to PTH (1-34). Confluent cell aggregates spontaneously formed mineralized nodules within 4-7 days, in the absence of inducers. These observations suggest that the primary spongiosa cells recapitulates the differentiation process in vitro in an accelerated fashion and may serve as a useful model to study osteoblast differentiation.

Open Innovation for Phenotypic Drug Discovery: The PD2 Assay Panel

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target “agnostic” fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD2 assay panel. Analysis of PD2 submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD2 have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD2, may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase.

Studies on a platelet‐derived growth factor (PDGF) responsive osteosarcoma cell line, MG‐63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3‐kinase inhibitors on serum‐stimulated cell proliferation and PDGF‐stimulated DNA replication, actin rearrangements, or Ptdlns 3‐kinase activity. In a dose‐dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2‐(4‐morpholinyl)‐8‐phenyl‐4H‐1‐benzopyran‐4‐one), inhibited serum‐stimulated MG‐63 cell proliferation. The mitogenic effects of PDGF on MG‐63 cells, as determined by incorporation of [3H]‐thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG‐63 cells stimulated by PDGF form distinct actin‐rich, finger‐like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3‐phosphate in PDGF‐stimulated MG‐63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3‐kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3‐kinase inhibitors on serum‐ and PDGF‐stimulated MG‐63 cell proliferation and PDGF‐stimulated morphological changes and suggest a greater role for Ptdlns 3‐kinase in these processes. J. Cell. Biochem. 64:182–195.

Parathyroid hormone (hPTH 1–38) stimulates the expression of UBP41, an ubiquitin‐specific protease, in bone

Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT‐PCR) and screened for genes, which are differentially expressed in osteoblast‐enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1–38) (8 μg/100 g). We found and cloned one full‐length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin‐specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8–4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1–38) exposure in both metaphyseal (6‐ to 8‐fold) and diaphyseal (3‐fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1–38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1–31), which stimulates intracellular cAMP, and PTHrP (1–34) both induced UBP41 mRNA expression; whereas PTH analogs (3–34) and (7–34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast‐like cell lines UMR106, ROS17/2.8, and BALC. PTH (1–38) treatment induced UPB41 expression (3.6‐ to 13‐fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4‐, 4.5‐, and 2.4‐fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin‐specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half‐life or localization of essential proteins are modified to maintain bone homeostasis. J. Cell. Biochem. 85: 229–242, 2002.

The expression of the nuclear matrix proteins NuMA, topoisomerase II-α, and -β in bone and osseous cell culture: Regulation by parathyroid hormone

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-α, and -β, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the α-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-α, and II-β changes during osteoblast development or with PTH treatment. NuMA and topo II-β were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-α. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-α-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-α expression.

Transcriptomic Analysis of Induced Pluripotent Stem Cells Derived from Patients with Bipolar Disorder from an Old Order Amish Pedigree.

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs, NPs and neurons matured in culture for either 2 weeks (termed early neurons, E) or 4 weeks (termed late neurons, L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate, enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically, 328 genes were differentially expressed between BPD and control L neurons including GAD1, glutamate decarboxylase 1 (2.5 fold) and SCN4B, the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology, GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism, protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD.

Calcitonin responsiveness and receptor expression in porcine and murine osteoclasts: a comparative study

The presence of the calcitonin (CT) receptor is a distinguishing characteristic of osteoclasts; however, species variability exists with respect to functional responsiveness to CT. In the present study, CT responsiveness and temporal expression of the CT receptor in differentiating cultures of porcine osteoclasts was examined and compared to murine osteoclasts. In vitro porcine osteoclast differentiation was evaluated using bone marrow cultures from neonatal pigs. Murine osteoclast differentiation was studied using cocultures of murine bone marrow and BALC cells, a calvarial-derived cell line. In the presence of 1,25 (OH)2D3, a time-dependent increase in osteoclast differentiation was observed in porcine and murine cultures. Salmon CT (sCT) and porcine CT (pCT) inhibited 1,25 (OH)2D3-stimulated porcine osteoclast differentiation at 10(-8) and 10(-7) mol/L (60% with 10(-7) mol/L sCT and 85% inhibition with 10(-7) mol/L pCT). Treatment of murine cocultures with sCT (10(-17)-10(-7) mol/L) resulted in a concentration-dependent decrease in osteoclast differentiation with a maximal inhibition of 70%. Osteoclast differentiation was inhibited in a concentration-dependent manner by recombinant human transforming growth factor-beta1 (rhTGF-beta1) in both species. The effects of CT on resorption lacunae formation were determined by culturing in vitro generated porcine or murine osteoclasts on bovine cortical bone slices for 18 h in the presence or absence of CT. With both porcine and murine osteoclasts, a concentration-dependent decrease in resorption lacunae formation was observed between 10(-13) and 10(-7) mol/L sCT with the highest concentrations completely abolishing resorption. However, pCT only inhibited porcine osteoclastic resorption at 10(-7) mol/L. CT receptor messenger ribonucleic acid (mRNA) expression was determined at different time points during in vitro osteoclast differentiation. In porcine cultures, expression of CT receptor mRNA correlated with the presence of osteoclasts. In murine cocultures, mRNA for the CT receptor was observed at each time point examined and was independent of the presence of multinucleated osteoclasts. Thus, porcine and murine differentiating osteoclast cultures express CT receptor mRNA; however, receptor expression correlates with osteoclast formation only in the porcine cultures. In summary, porcine and murine osteoclasts express CT receptor mRNA and functional responsiveness to CT. These findings suggest that the effects of sCT on osteoclast resorption are similar in murine and porcine cells, but that sCT is a less potent inhibitor of porcine than murine osteoclast differentiation.