Reduction by strontium of the bone marrow adiposity in mice and repression of the adipogenic commitment of multipotent C3H10T1/2 cells.

Multipotent mesenchymal cells (MMCs) differentiate into osteoblasts or adipocytes through RUNX2 and PPARγ2, respectively. Strontium ranelate has been shown to promote osteoblastogenesis and prevent adipogenesis in long-term experiments using MMCs. The present study involved in-vitro and in-vivo investigations of whether Sr might first be an inhibitor of adipogenesis, thus explaining late osteoblastogenesis. It was established in vivo that Sr reduces adipogenesis in mice treated only for 3 weeks with a 6 mmol/kg/day dose of Sr while the trabecular bone volume is increased. In order to decipher molecular mechanisms during inhibition of adipogenesis, we used murine MMCs C3H10T1/2 cultured under adipogenic conditions (AD) and treated Sr of a concentration up to 3 mM. It was shown that early on (day 1), Sr dose-dependently reduced PPARγ2 and CEBPα mRNA without affecting the RUNX2 gene expression whereas it repressed ALP mRNA. Later (day 5), PPARγ2 and CEBPα mRNA remained inhibited by Sr, preventing adipocyte lipid accumulation, while Runx2 and ALP mRNA were increased. Moreover, under the mentioned conditions, Sr was able to quickly induce the Cyclin D1 gene expression, proliferation and fibronectin fibrillogenesis, both involved in the inhibition of adipogenesis. The inhibition of the ERK pathway by U0126 blunted the Sr-induced PPARγ2 repression while restoring the lipid accumulation. These results demonstrated that Sr was capable of rapidly reducing adipogenesis by a selective PPARγ2 repression that can be explained by its ability to promote MMC proliferation.

Apatite content of collagen materials dose-dependently increases pre-osteoblastic cell deposition of a cement line-like matrix.

Bone matrix, mainly composed of type I collagen and apatite, is constantly modified during the bone remodeling process, which exposes bone cells to various proportions of mineralized collagen within bone structural units. Collagen-mineralized substrates have been shown to increase osteoblast activities. We hypothesized that such effects may be explained by a rapid secretion of specific growth factors and/or deposition of specific matrix proteins. Using MC3T3-E1 seeded for 32h on collagen substrates complexed with various apatite contents, we found that pre-osteoblasts in contact with mineralized collagen gave rise to a dose-dependent deposit of Vascular Endothelial Growth Factor-A (VEGF-A) and RGD-containing proteins such as osteopontin (OPN) and fibronectin (FN). This RGD-matrix deposition reinforced the cell adhesion to collagen-mineralized substrates. It was also observed that, on these substrates, this matrix was elaborated concomitantly to an increased cell migration, allowing a homogeneous coverage of the sample. This particular surface activation was probably done firstly to reinforce cell survival (VEGF-A) and adhesion (OPN, FN) and secondly to recruit and prepare surfaces for subsequent bone cell activity.

Physiological strains induce differentiation in human osteoblasts cultured on orthopaedic biomaterial.

We have developed an in vitro mechanical stretching model of osteoblastic cells cultured on metallic biomaterials in order to study the effects of mechanical strain on osteointegration of orthopaedic implants. Titanium alloy discs coated with alumina or hydroxyapatite were used as substrates. Three Dynacell devices were especially designed to apply cyclic strains on rigid biomaterials. The regimen (600 mu epsilon strains, 0.25Hz) was defined on the basis of physiological data and estimated deformation on hip stem prostheses. The performances of these apparatus were reproducible and provided controlled deformations. Human osteosarcoma cell line MG-63, human osteoblasts obtained from primary cultures and ROS 17/2.8 rat osteosarcoma cells were used as cell models. Cell behaviour was assessed in terms of growth and alkaline phosphatase (ALP) activity by in situ assays for two regimens: 15-min deformations repeated three times a day to mimic rehabilitation exercises and 24-h continuous deformations. We demonstrated that continuous deformation did not affect the growth and ALP activity of MG-63 cells, in contrast with sequential deformations which had no effect on cell number, but which stimulated ALP activity after 5 days of stretching. This sequential regimen can also modify the behaviour of human bone-derived cells resulting in increased proliferation after 5 days and stimulation of ALP activity after 15 days. ROS 17/2.8 rat osteosarcoma cells submitted to sequential deformations responded faster than other cell lines by increasing their ALP activity only after 1 day of stretching. Like MG-63 cells, proliferation of the ROS 17/2.8 rat osteosarcoma cell line was not affected by sequential deformations. This study suggests that short, repeated deformations defined to mimic rehabilitation exercises recommended after prostheses implantation are more likely to exert beneficial effects on implanted bone than continuous strains.

Production of matrix metalloproteinase 2 in fibroblast reaction to mechanical stress in a collagen gel.

The reaction of fibroblasts to mechanical forces generated by fibroblasts themselves in anchored collagen lattices was studied. Fibroblasts were cast in collagen gels. Free retracted gels (RG) were compared with stressed gels (SG) in 3-day and 14-day experiments. As previously described, SG showed an increase in protein, mainly collagen, biosynthesis. Matrix metalloproteinases (pro-MMP-2 and MMP-2) were studied by zymography. Certain cell membrane components, integrin alpha(2) and phosphatidylserine, were studied by flow cytometry with antibodies against the integrin alpha(2) subunit and with annexin V binding. Mechanical stress stimulated production of pro-MMP-2 both in the short-term (3-day) and the longer term (14-day) cultures. However, the pro-enzyme was not more activated and there was no difference in the amount of MMP-2 between RG and SG. There was only an increase with time under both conditions. The stressed fibroblasts reacted early with an increase in the integrin alpha(2) subunit, but the stimulated cells disappeared from the 14-day cultures. The number of cells measured in terms of the amount of DNA decreased between day 3 and day 14, mainly in the SG due to cytolysis. This cell stress was related to an alteration in the plasma membrane detected by the annexin V marker.

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins-termed secreted Frizzled-related proteins-that function as antagonists of Wnt signalling. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway.

Not bound by the law: legal disobedience in Israeli society.

The issue of whether there is a 'prima facie obligation to obey the law' has intrigued human society since the days of Socrates. However, most of the writings in this field have dealt with theoretical aspects of the issue, such as the boundaries of legal obedience and frameworks defining the circumstances under which a citizen is not obliged to obey the law. Very few studies have investigated the phenomenon of legal disobedience empirically. The current study is based on a survey of Israeli citizens belonging to three sectors of the population (Jews in the general population, Israeli Arabs, and orthodox Jewish students enrolled in religious yeshiva seminaries). Respondents' attitudes towards the judicial system, the rule of law, and the duty to obey state laws were examined by means of a questionnaire especially designed for the study. The findings point to gaps between the three groups: Compared to the Arab population and the yeshiva students, support for state laws and the rule of law was stronger among Jews in the general population and, conversely, belief in the supremacy of other laws (i.e. religious laws) over state laws and readiness to take the law into one's own hands were stronger among the Arabs and the yeshiva students, compared to the general Jewish population.

Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.

Retinol dehydrogenase (RDH), the enzyme that catalyzes the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment, was the first visual cycle enzymatic activity to be identified. Previous work has shown that this enzyme utilizes NADPH, shows a marked preference for all-trans-retinal over 11-cis-retinal, and is tightly associated with the outer segment membrane. This paper reports the identification of a novel member of the short chain dehydrogenase/reductase family, photoreceptor RDH (prRDH), using subtraction and normalization of retina cDNA, high throughput sequencing, and data base homology searches to detect retina-specific genes. Bovine and human prRDH are highly homologous and are most closely related to 17-beta-hydroxysteroid dehydrogenase 1. The enzymatic properties of recombinant bovine prRDH closely match those previously reported for RDH activity in crude bovine rod outer segment preparations. In situ hybridization and RNA blotting show that the PRRDH gene is expressed specifically in photoreceptor cells, and protein blotting and immunocytochemistry show that prRDH localizes exclusively to both rod and cone outer segments and that prRDH is tightly associated with outer segment membranes. Taken together, these data indicate that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment.

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (betaGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of betaGP supply were tested. We compared 10 mM betaGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where betaGP was added at day 0. Furthermore, 10 mM betaGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Interaction of a plasma-sprayed hydroxyapatite coating in contact with human osteoblasts and culture medium.

The loss of calcium from plasma-sprayed calcium phosphate ceramics (CPCs) on bioinert metal substrate (Ti-6Al-4V) immersed in cell culture medium with or without human osteoblast culture was measured. The ceramics were a CPC and a duplex system composed of a CPC layer on an alumina coating. The dissolution of calcium compounds was monitored by measuring the calcium leaked from the coatings into the culture medium in 15 days. Calcium was measured by flame photometry. The surfaces of the ceramics exposed to the culture medium and in contact with osteoblasts were analysed by X-ray diffraction (XRD). The dissolution process occurred in the first 6 days of contact, but the calcium released into the culture medium was only a small fraction of the calcium content of the coatings. The presence or absence of osteoblasts on the surface of the ceramics did not make significant difference for the calcium release. The XRD spectra of the ceramics before and after immersion and in contact with cells did not show a significant change in the compounds of the coatings.

A new secreted protein that binds to Wnt proteins and inhibits their activities.

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.

Cloning and characterization of a secreted frizzled-related protein that is expressed by the retinal pigment epithelium.

The Wnt/frizzled cell signaling pathway has been implicated in the determination of polarity in a number of systems, including the Drosophila retina. The vertebrate retina develops from an undifferentiated neuroepithelium into an organized and laminated structure that demonstrates a high degree of polarity at both the tissue and cellular levels. In the process of searching for molecules that are preferentially expressed by the vertebrate retinal pigment epithelium (RPE), we identified secreted frizzled-related protein 5 (SFRP5), a member of the SFRP family that appears to act by modulating Wnt signal transduction. SFRP5 is highly expressed by RPE cells, and is also expressed in the pancreas. Within the retina, the related molecule SFRP2 is expressed specifically by cells of the inner nuclear layer. Thus, photoreceptors are likely to be bathed by two opposing gradients of SFRP molecules. Consistent with SFRP5 's postulated role in modulating Wnt signaling in the retina, it inhibits the ability of Xwnt-8 mRNA to induce axis duplication in Xenopus embryos. The human SFRP5 gene consists of three coding exons and it maps to chromosome 10q24.1; human SFRP2 maps to 4q31.3. Based on the biology and complementary expression patterns of SFRP2 and SFRP5, we suggest that they may be involved in determining the polarity of photoreceptor, and perhaps other, cells in the retina.

Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.

CPG16, a novel protein serine/threonine kinase downstream of cAMP-dependent protein kinase.

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested, e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.

Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels.

Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 2-D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3-D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis-x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells.

Molecular genetics of human retinal disease.

The past decade has witnessed extraordinary progress in retinal disease gene identification, the analysis of animal and tissue culture models of disease processes, and the integration of this information with clinical observations and with retinal biochemistry and physiology. During this period over twenty retinal disease genes were identified and for many of these genes there are now significant insights into their role in disease. This review presents an overview of the basic and clinical biology of the retina, summarizes recent progress in understanding the molecular mechanisms of inherited retinal diseases, and offers an assessment of the role that genetics will play in the next phase of research in this area.

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms.

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

Morphological alteration of fibroblasts mechanically stressed in a collagen lattice.

The behavior of fibroblast-populated collagen lattices under mechanical stress was studied. Lattice retraction was blocked to allow the application of mechanical stress by contraction of the fibroblasts against the fixed ends of the lattice. The forces were modulated by varying the collagen/fibroblast ratio and the amount of collagen fibrils produced, by which the forces were transmitted. Transmission electron microscopy showed several disturbances of fibroblast ultrastructure, with empty and full vacuoles, lamellar bodies and signs of cytolysis. It is suggested that the morphological alterations in the fibroblasts may constitute a feedback reaction to the mechanical stress.

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors.

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.

A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.

Stargardt disease (STGD, also known as fundus flavimaculatus; FFM) is an autosomal recessive retinal disorder characterized by a juvenile-onset macular dystrophy, alterations of the peripheral retina, and subretinal deposition of lipofuscin-like material. A gene encoding an ATP-binding cassette (ABC) transporter was mapped to the 2-cM (centiMorgan) interval at 1p13-p21 previously shown by linkage analysis to harbour the STGD gene. This gene, ABCR, is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors, as detected by in situ hybridization. Mutational analysis of ABCR in STGD families revealed a total of 19 different mutations including homozygous mutations in two families with consanguineous parentage. These data indicate that ABCR is the causal gene of STGD/FFM.