S. E. Harris

Identification of the haematopoietic stem cell niche and control of the niche size.

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC ‘niche’—the in vivo regulatory microenvironment where HSCs reside—and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and β-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.

Light speed reduction to 17 metres per second in an ultracold atomic gas

Techniques that use quantum interference effects are being actively investigated to manipulate the optical properties of quantum systems. One such example is electromagnetically induced transparency, a quantum effect that permits the propagation of light pulses through an otherwise opaque medium. Here we report an experimental demonstration of electromagnetically induced transparency in an ultracold gas of sodium atoms, in which the optical pulses propagate at twenty million times slower than the speed of light in a vacuum. The gas is cooled to nanokelvin temperatures by laser and evaporative cooling. The quantum interference controlling the optical properties of the medium is set up by a ‘coupling’ laser beam propagating at a right angle to the pulsed ‘probe’ beam. At nanokelvin temperatures, the variation of refractive index with probe frequency can be made very steep. In conjunction with the high atomic density, this results in the exceptionally low light speeds observed. By cooling the cloud below the transition temperature for Bose–Einstein condensation (causing a macroscopic population of alkali atoms in the quantum ground state of the confining potential), we observe even lower pulse propagation velocities (17 m s−1) owing to the increased atom density. We report an inferred nonlinear refractive index of 0.18 cm2 W−1 and find that the system shows exceptionally large optical nonlinearities, which are of potential fundamental and technological interest for quantum optics.

Electromagnetically Induced Transparency

Electromagnetically induced transparency is a technique for eliminating the effect of a medium on a propagating beam of electromagnetic radiation. EIT may also be used, but under more limited conditions, to eliminate optical self‐focusing and defocusing and to improve the transmission of laser beams through inhomogeneous refracting gases and metal vapors, as figure 1 illustrates. The technique may be used to create large populations of coherently driven uniformly phased atoms, thereby making possible new types of optoelectronic devices.

A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases

Osteoblastic bone metastases are common in prostate and breast cancer patients, but mechanisms by which tumor cells stimulate new bone formation are unclear. We identified three breast cancer cell lines that cause osteoblastic metastases in a mouse model and secrete endothelin-1. Tumor-produced endothelin-1 stimulates new bone formation in vitro and osteoblastic metastases in vivo via the endothelin A receptor. Treatment with an orally active endothelin A receptor antagonist dramatically decreased bone metastases and tumor burden in mice inoculated with ZR-75-1 cells. Tumor-produced endothelin-1 may have a major role in the establishment of osteoblastic bone metastases, and endothelin A receptor blockade represents effective treatment.

Tunable optical parametric oscillators

This paper reviews progress on tunable optical parametric oscillators. Topics considered include: parametric amplification of Gaussian beams: threshold; tuning techniques, spectral output, and stability; saturation and power output; spontaneous parametric emission; nonlinear materials; and far infrared generation.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12

Since the bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily that induce the differentiation of mesenchymal precursor cells into the osteogenic cells, we identified the relevant signaling molecules responsible for mediating BMP-2 effects on mesenchymal precursor cells. BMP-2 induces osteoblastic differentiation of the pluripotent mesenchymal cell line C2C12 by increasing alkaline phosphatase activity and osteocalcin production. As recent studies have demonstrated that cytoplasmic Smad proteins are involved in TGF-β superfamily signaling, we plan to isolate the relevant Smad family members involved in osteoblastic differentiation. We identified human Smad5, which is highly homologous to Smad1. BMP-2 caused serine phosphorylation of Smad5 as well as Smad1. In contrast, TGF-β failed to cause serine phosphorylation of Smad1 and Smad5. We found Smad5 is directly activated by BMP type Ia or Ib receptors through physical association with these receptors. Following phosphorylation, Smad5 bound to DPC4, another Smad family member, and the complex was translocated to the nucleus. Overexpression of point-mutated Smad5 (G419S) or a C-terminal deletion mutant DPC4 (DPC4ΔC) blocked the induction of alkaline phosphatase activity, osteocalcin production, and Smad5-DPC4 signaling cascades upon BMP-2 treatment in C2C12 cells. These data suggest that activation of Smad5 and subsequent Smad5-DPC4 complex formation are key steps in the BMP signaling pathway, which mediates BMP-2-induced osteoblastic differentiation of the C2C12 mesenchymal cells.

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation

Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 µg/ml) and β-glycerophosphate (2.5, 5, or 10 mM βGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM βGP in the presence of 100 µg/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with βGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM βGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and βGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining only upon treatment with ascorbic acid and βGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing βGP, there was a statistically significant (P ≤ 0.02) dose-dependent increase in the mineral-to-matrix ratio (0.56 ± 0.16, 1.00 ± 0.32, and 2.46 ± 0.76, for 2.5, 5, and 10 mM βGP, respectively). The mean carbonate-to-phosphate ratios of the FRC cultures were 0.015, 0.012, and 0.008, in order of increasing βGP concentration, compared with rat calvaria values of 0.009–0.017. The 2T3 cells treated with BMP-2 also made bonelike crystals, similar to those observed in FRC cultures. In contrast, the cultures of von Kossa positive MC3T3-E1 cells did not display a significant amount of mineral (maximum mineral-to-matrix ratio was 0.4). Thus, although the von Kossa stainings of FRC, 2T3, and MC3T3-E1 were very similar, FTIR analysis indicated that calcium phosphate mineral was not present in the MC3T3 cultures. By EM, the mineral in FRC cell cultures and 2T3 cultures was generally associated with collagen, whereas rare or sparse dystrophic mineralization of unknown chemical origin was evident in the MC3T3-E1 cultures. These studies demonstrate that von Kossa staining alone is not appropriate for the identification and quantitation of bonelike mineral and, hence, other techniques such as X-ray diffraction, EM, or FTIR should be utilized to verify the presence and quality of calcium phosphate phases.

MLO-Y4 osteocyte-like cells support osteoclast formation and activation

Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte‐like cell line called MLO‐Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO‐Y4 cells support osteoclast formation in the absence of 1,25‐dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO‐Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200–1000 MLO‐Y4 cells per 0.75‐cm2 well. No osteoclast formation was observed with 2T3, OCT‐1, or MC3T3‐E1 osteoblast cells (1000 cells/well). Conditioned media from the MLO‐Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2‐week‐old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO‐Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony‐stimulating factor (M‐CSF), RANKL, and osteoprotegerin (OPG). MLO‐Y4 cells express and secrete large amounts of M‐CSF. MLO‐Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO‐Y4 cells compared with the other cell types. RANK‐Fc and OPG‐Fc blocked the formation of osteoclasts by MLO‐Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M‐CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes.