Peter V. Hauschka

Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment

Osteoblasts and endothelium constitute functional niches that support haematopoietic stem cells in mammalian bone marrow. Adult bone marrow also contains adipocytes, the number of which correlates inversely with the haematopoietic activity of the marrow. Fatty infiltration of haematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia. To explore whether adipocytes influence haematopoiesis or simply fill marrow space, we compared the haematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. Here we show, by flow cytometry, colony-forming activity and competitive repopulation assay, that haematopoietic stem cells and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 ‘fatless’ mice, which are genetically incapable of forming adipocytes, and in mice treated with the peroxisome proliferator-activated receptor-γ inhibitor bisphenol A diglycidyl ether, which inhibits adipogenesis, marrow engraftment after irradiation is accelerated relative to wild-type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone-marrow microenvironment, and indicate that antagonizing marrow adipogenesis may enhance haematopoietic recovery in clinical bone-marrow transplantation.

Osteocalcin: isolation, characterization, and detection.

Publisher Summary This chapter discusses the isolation, characterization, and detection of osteocalcin. Osteocalcin is a small protein (5800 daltons) comprising 10-20% of the noncollagenous protein in the bone. Because osteocalcin is tightly adsorbed to the hydroxyapatite mineral phase of the bone, thorough extraction is best achieved by dissolving the mineral of finely pulverized bone. Hydroxyapatite is soluble in neutral 0.5M ethylenediaminetetraacetic acid (EDTA), as well as in a variety of mineral and organic acids. Uniformly high yields of 1-2 mg of osteocalcin per gram of dry bone are obtained with EDTA, whereas acid procedures often leave 10-25% of the protein behind, presumably because of precipitation during excursions through the osteocalcin isoelectric point. The use of radioimmunoassay for the measurement of osteocalcin offers the advantages of specificity, sensitivity, and technical simplicity. The method can easily detect nanogram quantities of the protein in bone extracts, cell cultures, and serum. The assay is based on the competition of the radioactively labeled antigen and an identical nonlabeled antigen for binding to a specific antibody. The amount of labeled antigen bound to the antibody is inversely proportional to the amount of unlabeled antigen present in the system.

Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone‐forming osteoblasts and bone‐resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen‐induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre‐osteoclasts. We have characterized a cell‐type‐specific hormone‐inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor‐alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre‐osteoclasts by the same osteoblast‐dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre‐osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

Carboxylated calcium-binding proteins and vitamin K.

FIFTY years ago, Heinrik Dam, while a doctoral student under Rudolf Schonheimer at the University of Freiberg, was investigating cholesterol bio-synthesis in chicks. He prepared fat-free diets by e...

Quantitative determination of γ-carboxyglutamic acid in proteins

Abstract Methods for alkaline hydrolysis of proteins and chromatographic resolution and quantitation of the calcium-binding amino acid γ-carboxyglutamate (Gla) are presented. Gla is extremely acid labile and totally converted to glutamic acid during acid hydrolysis. Under the recommended hydrolysis conditions of 2 m KOH at 110°C for 22 hr, the recovery of Gla is quantitative. The ninhydrin color yield for Gla on our particular amino acid analyzer is only 39% of that for glutamic acid; this factor is readily obtained from the ratio of peak areas after acid conversion of Gla to glutamic acid.

Calcium regulates the PI3K-Akt pathway in stretched osteoblasts

Mechanical loading plays a vital role in maintaining bone architecture. The process by which osteoblasts convert mechanical signals into biochemical responses leading to bone remodeling is not fully understood. The earliest cellular response detected in mechanically stimulated osteoblasts is an increase in intracellular calcium concentration ([Ca2+]i). In this study, we used the clonal mouse osteoblast cell line MC3T3‐E1 to show that uniaxial cyclic stretch induces: (1) an immediate increase in [Ca2+]i, and (2) the phosphorylation of critical osteoblast proteins that are implicated in cell proliferation, gene regulation, and cell survival. Our data suggest that cyclic stretch activates the phosphoinositide 3‐kinase (PI3K) pathway including: PI3K, Akt, FKHR, and AFX. Moreover, cyclic stretch also causes the phosphorylation of stress‐activated protein kinase/c‐Jun N‐terminal kinase. Attenuation in the level of phosphorylation of these proteins was observed by stretching cells in Ca2+‐free medium, using intra‐ (BAPTA‐AM) and extracellular (BAPTA) calcium chelators, or gadolinium, suggesting that influx of extracellular calcium plays a significant role in the early response of osteoblasts to mechanical stimuli.

Nitric oxide acts in conjunction with proinflammatory cytokines to promote cell death in osteoblasts.

Proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), and interleukin‐1β are known modulators of bone remodeling in vitro and in vivo. The same cytokines induce the production of nitric oxide (NO) in various cell types, including osteoblasts and osteoclasts, and NO has recently been implicated in the regulation of bone resorption. We investigated the relationship between NO levels and cell viability in MC3T3‐E1, a well‐characterized osteoblastic cell line. NO donors at high concentrations (≥0.5 mM) produce a significant cytotoxic effect over a 48 h period. Various combinations of the three cytokines strongly promote endogenous NO production, and high NO levels are correlated with the loss of cell viability. Although TNF‐α produces NO‐independent cytotoxicity, NO greatly enhances this cytotoxic effect. Human and mouse TNF‐α differ in their cytotoxic effects, and human TNF‐α induces lower levels of NO production. In cocultures of RAW 264.7 mouse macrophages stimulated with lipopolysaccharide and IFN‐γ, and untreated MC3T3‐E1 osteoblasts, addition of anti–TNF‐α antibody and inhibition of NO synthesis have additive, protective effects on osteoblast viability. NO cytotoxicity involves an apoptotic mechanism. Our results underline the importance of NO and TNF‐α as cytotoxic mediators in the osseous microenvironment and might explain the observed deficiency of bone formation in inflammatory sites.

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1β and TNF-α were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1β and TNF-α were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)2-vitamin D3, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF-α stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1β and TNF-α failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1β and l nM TNF-α) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.

Vitamin K-dependent γ-carboxyglutamic acid formation by kidney microsomes in vitro

Abstract γ-Carboxyglutamic acid has been identified as a constituent of renal tissue in chicken, rat, and rabbit and is depressed by vitamin K-deficiency or dicoumarol diets. Thorough perfusion of rat and rabbit kidneys to remove blood contamination does not remove the γ-carboxyglutamate containing protein(s), which appear to be localized in the cortex. Incubation of kidney microsomes with [14C]NaHCO3in vitro results in the post-translational formation of protein bound [ 14 C]-γ-carboxyglutamic acid. Incorporation is stimulated 1.6- to 34-fold by addition of the active vitamin K 2-methyl, 3-farnesyl, 1,4-naphthoquinone. About 80% of incorporated, non-dialyzable 14C is situated in the γ-carboxyl group of γ-carboxyglutamic acid.

VEGF enhancement of osteoclast survival and bone resorption involves VEGF receptor-2 signaling and β3-integrin

VEGF dependent angiogenesis is required for normal bone development and has been implicated in cancer metastasis to bone. These processes, while dependent on osteoclastic bone resorption, are reportedly mediated by endothelial cells, stromal osteoblasts, chondrocytes, and/or tumor cells. We demonstrate here that VEGF treatment of purified murine bone marrow osteoclast precursors directly enhances their survival, differentiation into mature osteoclasts, and resorptive activity. The actions of VEGF on mature osteoclasts principally involve the receptor VEGFR2 (Flk1, KDR), and the receptor signaling utilizes both the PI3-kinase-->Akt and MEK-->ERK pathways. Increased osteoclast survival and resorptive activity is correlated with VEGF-dependent phosphorylation of multiple downstream targets of activated Akt [glycogen synthase kinase, GSK-3beta; forkhead transcription factor, FKHR; and the Bcl-2 antagonist of cell death, Bad (Ser136)] and activated ERK1/2 [ribosomal S6 kinase, p90RSK; and Bad (Ser112)]. Expression of the VEGFR2 gene increases 20-fold during the 6 day in vitro differentiation of mature osteoclasts from mononuclear precursors, while alternate receptors VEGFR1 and neuropilin-1, decrease 30- and 3-fold respectively. Additionally, VEGF enhancement of osteoclast survival is diminished in cells prepared from beta3 integrin-deficient mice, thus associating VEGF signaling in osteoclasts with their attachment to extracellular matrix. Our results indicate that VEGF directly targets osteoclasts, thereby playing a novel role in bone development, angiogenesis, and tumor metastasis.