A. Ian Cassady

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL‐Induced Signaling Pathways and Reactive Oxygen Species Production

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase‐3 activity, cytosolic Ca2+, and RANKL‐induced activation of NF‐κB and AP‐1 activities.

Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

Background Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. Principal Findings Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. Conclusion Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

The generation and properties of human macrophage populations from hemopoietic stem cells

Information about the development and function of human macrophage lineage populations, such as osteoclasts, is limited because of the lack of defined in vitro systems for their large‐scale generation. Two M‐CSF‐containing cytokine cocktails were found under serum‐free conditions to expand dramatically and to differentiate over time human CD34+ hemopoietic stem cells into nonadherent and adherent macrophage populations. These populations exhibited increasing degrees of maturity over a 3‐week period characterized by morphology, surface marker expression (CD11b, CD86, CD64, CD14, and c‐Fms), phagocytic function, and gene‐expression profiling using quantitative PCR and microarray analysis (principal component analysis, k‐means clustering, and gene ontology classification). As assessed by the last criterion, the adherent population obtained at 3 weeks from the one protocol tested had high similarity to the well‐studied peripheral blood monocyte‐derived macrophages. The one population tested could be induced to differentiate into osteoclasts in the presence of M‐CSF and receptor activator of NF‐κB ligand, as judged by morphology, gene expression, and bone‐resorbing ability. In addition to the large numbers of macrophage lineage cells able to be produced, this replicating system may be suitable for the molecular analysis of macrophage lineage commitment and progression and for gene targeting and delivery.

The resistance of macrophage-like tumour cell lines to growth inhibition by lipopolysaccharide and pertussis toxin.

Summary. The process of tumorigenesis is frequently associated with resistance to growth inhibition by physiological regulators of normal cells. Murine macrophage‐like cell lines BAC1.2F5, RAW264, J774.1A and PU5/1.8 were resistant to growth inhibition by bacterial lipopolysaccharide (LPS) and pertussis toxin, agents that blocked growth of primary bone marrow‐derived macrophages (BMDM) in the presence of macrophage colony‐stimulating factor (CSF‐1). The resistance of the CSF‐1‐dependent cell line BAC1.2F5 to growth inhibition by pertussis toxin argues against the possibility that pertussis toxin‐sensitive G proteins are essential for the pathway of growth stimulation by CSF‐1. Conversely, these data add further weight to the argument that LPS mediates some of its biological activities by mimicking the action of pertussis toxin and inhibiting G protein function. The resistance of cell lines to LPS and pertussis toxin was not correlated with any alteration in the expression of mRNA encoding any of three pertussis‐toxin sensitive G protein α subunits. The pattern of G protein expression was consistent between primary cells and tumour cells, suggesting that this is a differentiation marker. In particular, Giα2 mRNA was expressed at remarkably high levels in all of the cells. The specificity of LPS resistance was investigated by studying down‐regulation of CSF‐1 binding and induction of protooncogene c‐fos and tumour necrosis factor (TNF) mRNA. BAC1.2F5 cells were LPS‐resistant in each of these assays. In CSF‐1 binding. RAW264 and J774.1A responded in the same way as bone marrow‐derived macrophages but required higher doses of LPS, whereas c‐fos and TNF mRNA were induced in these cells at concentrations that did not inhibit growth.

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

BackgroundTartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.FindingsA new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine.ConlusionThe gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

Transgenic Overexpression of Tartrate-Resistant Acid Phosphatase Is Associated with Induction of Osteoblast Gene Expression and Increased Cortical Bone Mineral Content and Density

Bone remodeling is a central event in the maintenance of skeletal tissue, and involves cycles of resorption followed by the formation of bone tissue. The activity of osteoclasts and osteoblasts during these cycles is tightly regulated by systemic and local factors coupling the action of these cells. Tartrate-resistant acid phosphatase (TRAP) is predominantly expressed in bone by osteoclasts but has also been detected in osteoblasts and osteocytes. Moreover, TRAP can stimulate the differentiation of mesenchymal lineage cells, i.e. progenitors of osteoblasts and adipocytes. In order to further explore the effects of TRAP on bone turnover, the structural and molecular phenotypes of osteoclasts and osteoblasts were assessed in TRAP-overexpressing transgenic mice. Transgenic mice of both sexes display increased cortical bone mineral content and density, which cannot be accounted for by decreased bone resorption since osteoclast numbers and resorptive activity do not differ from wild-type mice. Examination of the osteoblast phenotype revealed that markers of bone formation, i.e. procollagen type I N-terminal propeptides, and osteoblast lineage markers as well as the TRAP 1B mRNA transcript are increased in TRAP-overexpressing mice. Expression of the osteoclast-selective TRAP 1C mRNA is not increased in TRAP transgenic mice. Elevated expression of TRAP mRNA and protein were detected in osteoblasts, osteocytes and in the bone matrix of TRAP transgenic mice, suggesting that TRAP overexpression in osteoblast lineage cells is associated with increased cortical bone mineral content and density. The data presented here support the hypothesis that TRAP overexpression in the osteoblastic cell lineage stimulates the differentiation and/or activation of these cells.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1

Abstract. Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3′ UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Mapping and characterization of the eukaryotic early pregnancy factor/chaperonin 10 gene family

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.