Lisa J. Robinson

Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.

ACTH protects against glucocorticoid-induced osteonecrosis of bone.

We report that adrenocorticotropic hormone (ACTH) protects against osteonecrosis of the femoral head induced by depot methylprednisolone acetate (depomedrol). This therapeutic response likely arises from enhanced osteoblastic support and the stimulation of VEGF by ACTH; the latter is largely responsible for maintaining the fine vascular network that surrounds highly remodeling bone. We suggest examining the efficacy of ACTH in preventing human osteonecrosis, a devastating complication of glucocorticoid therapy.

FSH-Receptor Isoforms and FSH-dependent Gene Transcription in Human Monocytes and Osteoclasts

Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8-10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes and osteoclasts transcribed TNFalpha in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2h in 25ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFalpha and TSG-6, increased 2- to 3-fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms.

Estrogen Inhibits RANKL-stimulated Osteoclastic Differentiation of Human Monocytes through Estrogen and RANKL-regulated Interaction of Estrogen Receptor-α with BCAR1 and Traf6

The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at approximately 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-beta-estradiol. Estrogen receptor-alpha (ERalpha) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERalpha. However, ERalpha was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERalpha in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (approximately 5 min) estrogen-dependent formation of ERalpha-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-kappaB activity, precipitated with this complex. Reduction of NF-kappaB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IkappaB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERalpha.

Loss of DNA Polymerase ζ Enhances Spontaneous Tumorigenesis

Mammalian genomes encode at least 15 distinct DNA polymerases, functioning as specialists in DNA replication, DNA repair, recombination, or bypass of DNA damage. Although the DNA polymerase zeta (polzeta) catalytic subunit REV3L is important in defense against genotoxins, little is known of its biological function. This is because REV3L is essential during embryogenesis, unlike other translesion DNA polymerases. Outstanding questions include whether any adult cells are viable in the absence of polzeta and whether polzeta status influences tumorigenesis. REV3L-deficient cells have properties that could influence the development of neoplasia in opposing ways: markedly reduced damage-induced point mutagenesis and extensive chromosome instability. To answer these questions, Rev3L was conditionally deleted from tissues of adult mice using MMTV-Cre. Loss of REV3L was tolerated in epithelial tissues but not in the hematopoietic lineage. Thymic lymphomas in Tp53(-/-) Rev3L conditional mice occurred with decreased latency and higher incidence. The lymphomas were populated predominantly by Rev3L-null T cells, showing that loss of Rev3L can promote tumorigenesis. Remarkably, the tumors were frequently oligoclonal, consistent with accelerated genetic changes in the absence of Rev3L. Mammary tumors could also arise from Rev3L-deleted cells in both Tp53(+/+) and Tp53(+/-) backgrounds. Mammary tumors in Tp53(+/-) mice deleting Rev3L formed months earlier than mammary tumors in Tp53(+/-) control mice. Prominent preneoplastic changes in glandular tissue adjacent to these tumors occurred only in mice deleting Rev3L and were associated with increased tumor multiplicity. Polzeta is the only specialized DNA polymerase yet identified that inhibits spontaneous tumor development.

Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Purpose: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). Experimental Design: We studied growth factor–dependent and growth factor–independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. Results: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at ∼1 × 10−9 mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at ∼1 × 10−6 mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI50 of 5 × 10−9 mol/L. Primary AML blast cells exhibited a growth inhibition of <1 × 10−6 mol/L. Cell lines that showed growth inhibition at ∼1 × 10−6 mol/L showed a G1 cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. Conclusions: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects. Clin Cancer Res; 16(4); 1149–58

The role of calcium release activated calcium channels in osteoclast differentiation.

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m‐CSF and the TNF‐family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca2+. Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m‐CSF and RANKL, revealing significant loss in both the expression and function of the required components of store‐operated Ca2+ entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4‐dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down‐regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states. J. Cell. Physiol. 226: 1082–1089, 2011.

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1−/− mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1−/− mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1−/− mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1−/− animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1−/− and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.

Tumor necrosis factor family receptors regulating bone turnover: new observations in osteoblastic and osteoclastic cell lines.

Abstract:  While the tumor necrosis factor (TNF) family members RANKL and TNF‐α are critical regulators of osteoclast formation, functions of other TNFs in bone are poorly understood. Here we consider the roles in regulating bone turnover of TNF receptors (TNF‐R) also expressed by osteoblasts and osteoblast precursors. TNF receptors in osteoblasts and preosteoblasts include TNFR1 (p55), DR3 (TNFR25), DR5 (TRAIL‐R2) and Fas, and possibly FN14 and DR4 (TRAIL‐R1). Osteoblasts also produce soluble TNF receptors, DcR2, osteoprotegerin, and sDR3; these bind the TNFs TRAIL, RANKL, TL1A, and Apo3L and block ligand effects on cell surface receptors. Activation of DR3 regulates osteoblast maturation and may control the decision to exit the pool of differentiation‐competent preosteoblasts. A major natural ligand for DR3, TL1A, is produced by vascular cells adjacent to differentiating osteoblasts and possibly by Fcγ‐stimulated osteoclast precursors. The activity of DR3 is regulated by osteoblast production of its soluble DR3 splice variant. Activation of TNFR1 or DR5 by TNF‐α or TRAIL may regulate osteoblast connectivity, which is important to bone turnover. When there is a source for Fas ligand, such as an inflammatory infiltrate, activation of Fas may lead to apoptosis of any bone cell. TNF receptors are thus implicated in multiple aspects of bone turnover.

Calcium and bone disease.

Calcium transport and calcium signaling are of basic importance in bone cells. Bone is the major store of calcium and a key regulatory organ for calcium homeostasis. Bone, in major part, responds to calcium-dependent signals from the parathyroids and via vitamin D metabolites, although bone retains direct response to extracellular calcium if parathyroid regulation is lost. Improved understanding of calcium transporters and calcium-regulated cellular processes has resulted from analysis of genetic defects, including several defects with low or high bone mass. Osteoblasts deposit calcium by mechanisms including phosphate and calcium transport with alkalinization to absorb acid created by mineral deposition; cartilage calcium mineralization occurs by passive diffusion and phosphate production. Calcium mobilization by osteoclasts is mediated by acid secretion. Both bone forming and bone resorbing cells use calcium signals as regulators of differentiation and activity. This has been studied in more detail in osteoclasts, where both osteoclast differentiation and motility are regulated by calcium.