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W4233722201 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins. https://doi.org/10.7554/eLife.05914.001 eLife digest The forces applied to the body during daily activities cause bones to be constantly remodeled, which is essential for keeping them healthy. In most adult organisms, new bone is created at the same rate at which old bone is destroyed. This means that overall bone mass remains the same. But, in diseases such as osteoporosis or bone cancer, bone is destroyed more rapidly than at which new bone is made. This leads to brittle bones that are more likely to fracture. Understanding how to increase the rate of bone renewal might therefore help scientists develop new treatments for bone diseases. Bone is created by cells called osteoblasts and destroyed by other cells called osteoclasts. Both of these types of cells develop from stem cells in the bone marrow. The activity of these cells is controlled by a number of factors, including the matrix of proteins that holds bone together. A group of proteins called galectins are known to act as a bridge between some of the matrix proteins and molecules on the surface of the cells. Vinik et al. took osteoblasts from a mouse skull, grew them in the laboratory, and then exposed them to a galectin protein called galectin-8. This made the osteoblasts release a protein called RANKL, which is known to boost osteoclast activity. When osteoblasts that had been exposed to galectin-8 were grown alongside bone marrow stem cells, more of the stem cells developed into the bone-destroying osteoclasts. Mice that were genetically engineered to produce more galectin-8 than normal mice develop brittle bones, despite also creating new bone at a higher rate than do normal mice. This is because osteoclast activity increases at a greater rate, resulting in an overall loss of bone in these animals. This is similar to what occurs in some individuals with osteoporosis. These experiments therefore suggest that galectin-8 plays an important role in bone remodeling and that it may be a potential target for drugs that treat diseases that weaken bones. https://doi.org/10.7554/eLife.05914.002 Introduction Bone is a dynamic tissue that constantly undergoes remodeling by osteoclast-mediated bone resorption and osteoblast-mediated bone formation (Eriksen, 2010; Nakahama, 2010; Raggatt and Partridge, 2010). In a rapidly growing and mature mammals, bone remodeling is positive or balanced, respectively, allowing for bone mass accrual and later for its maintenance. Negatively balanced bone remodeling is a hallmark of pathologies such as osteoporosis and cancerous osteolysis (Kozlow and Guise, 2005; Novack and Teitelbaum, 2008; Sturge et al., 2011). Skeletal tissues are composed largely of extracellular matrix (ECM). Fibrillar ECM proteins, predominately type I collagen in bone and type II collagen in cartilage, provide structural integrity and account for mechanical strength. The ECM of bone also contains matricellular proteins that primarily serve as biological modulators. Matricellular proteins interact with cell-surface receptors, such as integrins, the structural matrix, and soluble extracellular factors including growth factors and proteases (Bornstein and Sage, 2002). Through these multiple interactions, matricellular proteins modulate cell function and regulate the availability and activity of proteins sequestered in the matrix. Therefore, matricellular proteins contribute to skeletal development, homeostasis, and fracture healing (Alford and Hankenson, 2006). Galectins are a family of glycan-binding proteins secreted by a variety of cell types (Boscher et al., 2011; Di Lella et al., 2011). As such, they can act as biological cross-linkers for ECM proteins and cell-surface receptors (Elola et al., 2007). Indeed, certain galectins were reported to function as matricellular proteins (Troncoso et al., 2014). The minimal structures recognized by these lectins are β-galactosides displayed on the cell surface as part of more complex glycoconjugates (Di Lella et al., 2011). The prototype galectins (galectin-1, -2, -5, -7, -10, -11, -13, -14, and -15) exist as monomers or homodimers of one carbohydrate recognition domain (CRD). The tandem-repeat-type galectins (galectin-4, -6, -8, -9, and -12) harbor two distinct CRDs joined by a peptide linker. The chimera-type galectin-3 consists of a CRD connected to a polypeptide that can pentamerize upon binding to glycan ligands (Rabinovich and Vidal, 2011; Thiemann and Baum, 2011; Ledeen et al., 2012). Being secreted proteins, as well as proteins having intracellular roles, galectins affect a wide range of biological functions including regulation of cell adhesion, migration, cell growth, apoptosis, and autophagy (Rabinovich and Vidal, 2011; Thiemann and Baum, 2011; Thurston et al., 2012). Still, regulation of bone physiology by galectins has been addressed only to a limited extent. Galectin-9 has been shown to induce osteoblast differentiation initiated by coupling of CD44 to bone morphogenetic protein (BMP) receptors (Tanikawa et al., 2010), whereas GC-1 and GC-8, the chicken orthologs of galectin-1 and galectin-8, respectively, were shown to mediate the formation and patterning of pre-cartilage mesenchymal condensations in the developing limb of chicken (Bhat et al., 2011). To study the possible role of galectins as regulators of bone physiology, we focused upon galectin-8 (gal-8), initially cloned in our laboratory, which is a tandem-repeat-type galectin having two sugar-binding domains joined by a linker peptide (Hadari et al., 1995; Levy et al., 2006). Upon secretion, galectin-8 is equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell-surface integrins and ECM proteins (Hadari et al., 2000; Levy et al., 2001; Eshkar Sebban et al., 2007). Complex formation between galectin-8 and integrins triggers integrin-mediated signaling cascades (Levy et al., 2001, 2003) that affect cell growth, receptor trafficking, and metastatic potential (Boura-Halfon et al., 2003; Zick et al., 2004; Arbel-Goren et al., 2005; Reticker-Flynn et al., 2012). In this study, we show that galectin-8 regulates bone mass by inducing the secretion of the osteoclastogenic factor, receptor activator of NF-κB ligand (RANKL) (Hanada et al., 2010), from isolated osteoblasts in a cell autonomous manner. As a result, co-culture of galectin-8-treated osteoblasts with bone marrow cells increases their differentiation into active osteoclasts. These effects involve the binding of galectin-8 to the osteoblasts' urokinase plasminogen-activated receptor (uPAR); mannose receptor C, type 2 (MRC2); and the low-density lipoprotein receptor-related protein 1 (LRP1), and seem to be of physiological relevance because galectin-8 transgenic animals exhibit increased expression of RANKL, increased osteoclastogenic activity, and enhanced bone turnover that culminates in reduced bone mass. These data identify galectin-8 as a potential drug target for the prevention of diseases associated with excessive bone loss. Results Effects of galectin-8 on cultured osteoblasts To study the effects of galectin-8 on osteoblasts in culture, osteoblasts derived from calvaria of newborn CD1 mice were incubated with 50 nM galectin-8. As shown in Figure 1A, such treatment increased by sixfold the expression of RANKL in these cells by 4 hr and resulted in a 2.5-fold increase in secretion of soluble RANKL into the medium (Figure 1B) by 24 hr. Extended incubation with galectin-8, up to 6 days maintained the high levels of expression of RANKL (Figure 1C). Galectin-8 also had a moderate (30%) inhibitory effect on the expression of osteoprotegerin (OPG), a neutralizing decoy receptor of RANKL (Eriksen, 2010) (Figure 1D). As a result, there was an overall 10-fold decrease in the ratio of OPG/RANKL transcription in galectin-8-treated osteoblasts (Figure 1E). We could therefore conclude that galectin-8 increases the RANKL/OPG ratio in osteoblasts in a cell autonomous manner. We have previously shown that galectin-8 is secreted and localizes to the extracellular surface of cells (Hadari et al., 2000). To determine whether galectin-8, secreted from osteoblasts, exerts similar effects, the expression of this lectin in osteoblasts was silenced using siRNA. As a result, a reduction of 87% in the expression levels of galectin-8 was accompanied by a significant reduction of 33% in the expression levels of RANKL (Figure 1F). We could therefore conclude that galectin-8 derived from osteoblasts can mediate RANKL expression, along with other stimuli that induce RANKL. Figure 1 Download asset Open asset Effects of galectin-8 on RANKL and OPG expression in osteoblasts. Osteoblasts derived from calvaria of newborn mice were treated with 50 nM of galectin-8 for 4 hr (A, D); 24 hr (B); or for the indicated times (C). After treatments, RNA was extracted and qRT-PCR was conducted in order to quantify changes in expression of RANKL (A, C) or osteoprotegerin (OPG) (D). Actin served as a control for normalization purposes. The levels of soluble RANKL in the medium were quantified by ELISA (B). (E) OPG/RANKL expression ratio was calculated from the results of A and D. (F) Osteoblasts from the calvaria of newborn mice were grown in 12-well plates (5 × 104 cells per well). After 24 hr, cells were transfected with siRNA for galectin-8. Non-targeting siRNA (siNONT) served as the control. 96 hr thereafter, cells were harvested, RNA was extracted, and qRT-PCR was conducted to quantify changes in mRNA levels of galectin-8 and RANKL. The content of actin mRNA served as a control for normalization purposes. (G) Bone marrow cells were extracted and analyzed by flow cytometry for the surface expression of galectin-8. Cells were treated with TDG or sucrose (10 mM in PBS) or just PBS before fixation. Results shown are of a representative histogram of cell number vs florescence intensity of the secondary antibody (left) and quantitation of the averages florescence intensity of the secondary antibody in two experiments carried out in duplicates (right). Results are mean values ± SEM of five (F) or six independent experiments (A, D, E) or of two independent experiments carried out in duplicates (C, G) or triplicates (B) *p < 0.05, **p < 0.01. https://doi.org/10.7554/eLife.05914.003 To determine whether bone marrow cells can also secrete galectin-8, they were subjected to analysis by flow cytometry. This analysis revealed that indeed primary murine bone marrow cells express and secrete galectin-8 (Figure 1G). This surface-bound galectin-8 could be partially displaced by thiodigalactoside (TDG), which blocks lectin–carbohydrate interactions, but not by sucrose, suggesting that surface binding of secreted galectin-8 is mediated, at least in part, through protein–carbohydrate interactions. Effects of galectin-8 on RANKL expression in osteoblasts vs osteocytes Recent studies have implicated matrix-embedded osteocytes, rather than osteoblasts, in the control of osteoclast formation (Nakashima et al., 2011; Xiong et al., 2011). To determine which cell type serves as a target for galectin-8, calvariae from newborn mice were separated using sequential digestion (Nakashima et al., 2011) into osteoblast-rich fraction, expressing the osteoblastogenic marker KERA (keratocan) (Nakashima et al., 2011) (Figure 2A), and an osteocyte-enriched fraction, which is almost devoid of KERA-expressing cells (Paic et al., 2009), but expresses DMP1, an osteocyte marker (Bonewald, 2011). As expected, expression of DMP1 was enriched threefold in the kera(−) fraction, although a significant number of DMP1(+) cells were also present in the kera(+) fraction (Figure 2B). Basal RANKL expression was much higher in the Kera(+) osteoblasts-enriched fraction than in the osteocyte-enriched fraction (Figure 2C). Furthermore, the level of RANKL expression in galectin-8-treated cells was fivefold higher than in basal both in Kera(+) and in Kera(−) cells (Figure 2C). Given that the Kera(−) fraction was almost completely devoid of osteoblasts, these results support the conclusion that galectin-8 affects RANKL expression both in cultured osteoblasts and osteocytes, derived from calvaria of newborn mice. Figure 2 Download asset Open asset Effects of galectin-8 on osteoblast fractions isolated from calvaria of newborn mice. Osteoblasts were extracted from calvaria of newborn mice by five sequential incubations with collagenase-dispase solution. Osteoblasts derived from the different incubations were seeded for 24 hr. KERA (A) and DMP1 (B) expression, using qRT-PCR, were examined in fractions −2 and −5 that showed the highest and the lowest amount of KERA (designated kera+ and kera−), respectively. (C) Galectin-8 (50 nM) was added to osteoblasts from these cultures for 24 hr; RANKL expression was determined by qRT-PCR. Actin served as a control for normalization purposes. Results are mean values ± SEM of n = 8 (A), n = 6 (B), n = 7 (C). *p < 0.05, **p < 0.01. https://doi.org/10.7554/eLife.05914.004 Effects of galectin-8 on osteoclasts differentiation The increased expression of the osteoclastogenic factor RANKL in osteoblasts treated with galectin-8 prompted us to study the effects of this lectin on osteoclasts differentiation in culture. For this purpose, bone marrow cells were co-cultured with osteoblasts derived from calvaria of newborn mice. We could demonstrate (Figure 3A) that galectin-8, added to this co-culture, was equipotent to the osteoclastogenic factor PGE2 (Suda et al., 2004) in the induction of a ∼15-fold increase in osteoclast differentiation, as evident by the appearance of multinucleated TRAP+ cells. The effects of PGE2 and galectin-8 were additive to a certain extent, suggesting that they might act by somewhat different mechanisms. Very few differentiated osteoclasts appeared in untreated co-cultures. Furthermore, galectin-8 had no direct differentiation effect on osteoclasts, as addition of this lectin to naive bone marrow cells in the absence of osteoblasts did not result in osteoclasts differentiation (Figure 3A). To verify that RANKL indeed mediates the effects of galectin-8 on osteoclasts differentiation, its expression was silenced using siRNAs. As shown in Figure 3B, RANKL-siRNAs reduced its transcription in osteoblasts by 50%, and this was accompanied by a similar 50% reduction in the ability of galectin-8 to induce osteoclastogenesis in co-culture experiments (Figure 3C). These results support the conclusion that galectin-8 functions as an osteoclastogenic agent through its action as an inducer of RANKL expression in osteoblasts. Figure 3 Download asset Open asset Effects of galectin-8 on osteoclast differentiation. (A) Osteoblasts (OBL) derived from the calvaria of newborn mice were seeded in 24-well plates (4 × 104 cells/well). After reaching 60–70% confluence, murine bone marrow cells (BM) extracted from the femur and tibia of 6-week-old mice were added to the culture (2 × 106 cells/well), together with galectin-8 (50 nM), PGE2 (1 μM), or both. Galectin-8 and PGE2 were further added on every other day for 10 days. TRAP assay was performed, and active osteoclasts (multinucleated TRAP+ cells) were counted. Results are mean values ± SEM of three independent experiments carried out in duplicates. (B) Osteoblasts were seeded in 12-well plates (5 × 104 cells per well). After 24 hr, cells were transfected with siRNA to RANKL. Non-targeting siRNA served as a control. 72 hr thereafter cells were harvested, RNA was extracted, and qRT-PCR was performed in order to quantify changes in mRNA levels of RANKL. The content of actin mRNA served as a control for normalization purposes. (C) Osteoblasts were seeded as in A. After reaching 60–70% confluence, cells were transfected with the indicated siRNAs for 72 hr. Thereafter, murine bone marrow cells extracted from the femur and tibia bones of 6-week-old mice were added to the culture (2 × 106 cells/well). Galectin-8 (50 nM) was added on the first, fourth, and sixth days after addition of bone marrow. Active osteoclasts were counted as in A. Results are mean values ± SEM of three (A, B) and two (C) independent experiments each carried out in duplicates (*p < 0.05, **p < 0.01). https://doi.org/10.7554/eLife.05914.005 Signaling pathways triggered by galectin-8 in osteoblasts The signaling pathways that mediate the effects of galectin-8 on osteoblasts were explored next. We could demonstrate that treatment of osteoblasts, derived from calvaria of newborn mice, with soluble galectin-8 (50 nM, 4 hr), induced the phosphorylation of ERK and Akt, while inhibitors of these signaling pathways—PD98095 and wortmannin, respectively—inhibited these phosphorylations (Figure 4A). PD98095 inhibited the ability of galectin-8 to promote transcription of RANKL in osteoblasts, whereas inclusion of wortmannin had no such an effect (Figure 4B), suggesting that the effects of galectin-8 on RANKL gene transcription are mediated by the ERK signaling pathway. PD98095 also effectively inhibited the appearance of multinucleated TRAP+-differentiated osteoclasts, when bone marrow cells were co-cultured with osteoblasts in the presence of galectin-8 (Figure 4C), indicating that the ERK signaling pathway is involved in this process as well. Wortmannin was capable of eliciting a partial inhibitory effect on osteoclast differentiation (Figure 4C), suggesting that the PI3K/Akt pathway could act at a step downstream or independent of RANKL transcription. Figure 4 Download asset Open asset Signaling pathways activated by galectin-8. (A) Osteoblasts derived from calvaria of newborn mice were treated with 25 μM PD98095 or 1 μM wortmannin for 1 hr before adding galectin-8 (50 nM). After 4 hr, total proteins were extracted and analyzed by Western blotting using antibodies specific for the phosphorylated forms of ERK and Akt. Shown is a representative of three experiments. (B) Osteoblasts were treated with PD98095 (25 μM) or wortmannin (1 μM) for 1 hr before being treated with 50 nM galectin-8. After 24 hr, cells were removed from plates, RNA was extracted, and qRT-PCR was performed in order to quantify changes in RANKL transcription. Actin served as a control for normalization purposes. Results shown are mean values ± SEM of three independent experiments, each done in duplicates. (C) Osteoblasts were seeded in 24-well plates (4 × 104 cells/well). After reaching 60–70% confluence, murine bone marrow cells extracted from the femur and tibia of 6-week-old mice were added to the culture (2 × 106 cells/well). Galectin-8 (50 nM), PD98095 (25 μM), and wortmannin (1 μM) were added every other day for 10 days. Multinucleated TRAP+ cells were scored as differentiated osteoclasts. Results shown in (C) are mean values ± SEM of two independent experiments each carried out in duplicate. (*p < 0.05, **p < 0.01). https://doi.org/10.7554/eLife.05914.006 Receptors for galectin-8 in osteoblasts To identify osteoblast receptors that could mediate the effects of galectin-8, proteins extracted from calvaria of newborn rats were affinity purified over columns of immobilized GST-galectin-8 and were analyzed by mass spectrometry. Two proteins that specifically bound to the columns were of interest: LRP1 (low-density lipoprotein receptor-related protein 1) (Grey et al., 2004) and MRC2 (mannose receptor C, type 2) (Engelholm et al., 2009). Both LRP1 and MRC2 could be detected by staining of proteins that selectively bound to immobilized GST-galectin-8 (Figure 5A). Because LRP1 and MRC2 form complexes with the urokinase plasminogen activator receptor (uPAR) (Behrendt, 2004; Gonias et al., 2011), it was of interest to determine whether uPAR is also part of the proteins complex that binds galectin-8. Indeed, we could show by Western blotting that similar to LRP1 and MRC2, uPAR also selectively binds to immobilized galectin-8 (Figure 5B). Figure 5 Download asset Open asset Binding of proteins extracted from osteoblasts to GST-galectin-8. (A, B) Calvariae were isolated from newborn rats (A) or mice (B); homogenized, and proteins were extracted and incubated for 16 hr at 4°C with GST- or GST-gal-8-loaded beads. Next, the beads were washed in PBS+1% Triton X-100. Elution was performed with 0.5M lactose, and the eluted proteins were resolved by SDS-PAGE and were stained with GelCode (A). Relevant bands (marked with a rectangle) were excised, trypsinized, and subjected to analysis by mass spectrometry. Alternatively, the eluted proteins were resolved by SDS-PAGE and were transferred to nitrocellulose membrane for Western blotting with the indicated antibodies (B). Blots shown are representatives of four independent experiments with similar results. https://doi.org/10.7554/eLife.05914.007 To evaluate the possible physiological relevance of these galectin-8-binding partners, their siRNAs were introduced into osteoblasts from calvaria of newborn mice. Transcription of MRC2, LRP1, and uPAR in osteoblasts was reduced >60–80% by their corresponding siRNAs (Figure 6A–C). Silencing of MRC2 inhibited (65%) the effects of galectin-8 on RANKL transcription in osteoblasts (Figure 6D) and inhibited by 40% the ability of these osteoblasts to promote osteoclastogenesis when co-cultured with bone marrow cells (Figure 6E). Similarly, siRNAs to uPAR effectively reduced (50%) the ability of galectin-8 to stimulate expression of RANKL (Figure 6F), suggesting that uPAR, like MRC2, mediates at least in part, the stimulatory effects of galectin-8 on RANKL transcription and osteoclastogenesis. By contrast, silencing of LRP1 significantly increased ∼2.5-fold the effects of galectin-8 on RANKL transcription (Figure 6G), suggesting that LRP1 could function as an inhibitory decoy receptor for galectin-8, impeding its ability to promote expression of RANKL. Figure 6 Download asset Open asset Effects of silencing of MRC2, LRP1, and uPAR on the mode of action of galectin-8. (A–D, F, G) Osteoblasts from calvaria of newborn mice were grown in 12-well plates (5 × 104 cells per well). After 24 hr, cells were transfected with the indicated siRNAs. Non-targeting siRNA served as control. 48–72 hr thereafter, galectin-8 (50 nM) was added for another 24 hr. Cells were then harvested, RNA was extracted, and qRT-PCR was conducted to quantify changes in mRNA levels of MRC2 (A), LRP1 (B), uPAR (C), and RANKL (D, F, G). The content of actin mRNA served as a control for normalization purposes. Results shown are mean values ± SEM of (n = 5 [A–C, F, G]; n = 3 [D]) **p < 0.01 vs control. (E) Osteoblasts were seeded in 24-well plates (4 × 104 cells/well). After reaching 60–70% confluence, cells were transfected with the indicated siRNAs. After 72 hr, murine bone marrow cells extracted from the femur and tibia of 6-week-old mice were added to the culture (2 × 106 cells/well). Galectin-8 (50 nM) was added on the first, fourth, and sixth days after addition of the bone marrow. TRAP assay was performed, and multinucleated TRAP+ cells were counted. Results are mean values ± SEM of triplicate measurements repeated in two independent experiments **p < 0.01 vs control. https://doi.org/10.7554/eLife.05914.008 Characteristics of transgenic mice overexpressing galectin-8 (gal-8 Tg) To further assess the physiological significance of the above findings, transgenic mice that overexpress galectin-8 were generated as described under ‘Materials and methods’. These mice express Myc-tagged galectin-8 controlled by the chicken beta-actin promoter that did not include a leader sequence. The insert was localized to chromosome 2, in a region free of genes or other known genomic features. Homozygous mice were used in this study. Mice were born at normal size and expressed no apparent deformity. They were fertile and propagated at a normal Mendelian distribution. Age- and sex-matched mice served as the control group. Immunohistochemical staining of bone sections and quantitative reverse transcriptase polymerase chain reaction quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis revealed that galectin-8 expression was increased ∼sixfold in osteoblasts derived from calvariae of newborn mice, whereas a ∼10-fold increase was observed in osteoblasts derived from long bones of 16-week-old transgenic mice (Tg) mice, when compared with the control wild-type (WT) mice (Figure 7A). Decoration with anti-galectin-8 antibodies of decalcified sections of tibia from 12-week-old WT mice and gal-8 Tg mice confirmed these results. A marked increase in anti-galectin-8 antibody binding was observed in sections of tibia derived from gal-8 Tg mice when compared with WT controls (Figure 7B). Figure 7 Download asset Open asset Expression of galectin-8 in femur and tibia of gal-8 Tg mice. (A) RNA was extracted from osteoblasts derived either from calvaria of newborn mice (n = 7) (left) or from the femur and tibia of 14-week-old (n = 5) (right) wild-type (WT) and gal-8 Tg mice. qRT-PCR was conducted using primers for galectin-8 or actin (control). Result shown are mean ± SEM (**p < 0.01). (B) Tibia was removed from 12-week-old WT (left) and gal-8 Tg mice (right). Bones were decalcified and fixed in paraffin blocks. Sections were cut and stained with anti-galectin-8 antibody (red) and DAPI (blue). https://doi.org/10.7554/eLife.05914.009 Effects of overexpression of galectin-8 on bone morphology To determine whether the osteoclastogenic activity of galectin-8 affects bone morphology, indices of tibial bone mass and architecture of WT and Tg mice were determined by micro-computed tomography (μCT) scans both in vivo and in vitro. In vitro μCT of the proximal region of the tibia of 16-week-old mice revealed bone osteopenia in the gal-8 Tg mice (Figure 8). This was characterized by a 57% decrease in Tb.N and 62% decrease in BV/TV ratio (Table 1). As a consequence, Tb.Sp was significantly higher (2.8-fold) in the Tg animals (Table 1). No change in Tb.Th was observed (not shown). A significant reduction (32%) in bone mineral density (BMD) of the Tg mice was observed as well (Table 1). Qualitatively, similar changes were also detected by in vivo μCT scans (Table 1). Same changes were also evident upon scanning of the distal region of the tibia (not shown). Figure 8 Download asset Open asset MicroCT scans of tibia proximal regions. In vivo and in vitro μCT scans were performed on 14-week-old WT and Tg mice, or on tibial bones removed from 16-week-old WT and Tg mice, respectively. Representative pictures show the proximal region of the tibia (for in vivo CT) or a region of interest within the trabecular bone of the tibia metaphysis (for in vitro CT). The position of Trabeculae is indicated by arrows. https://doi.org/10.7554/eLife.05914.010 Table 1 Analysis and stereological parameters of tibia proximal region in WT and gal-8 Tg mice https://doi.org/10.7554/eLife.05914.011 In vivo CTIn vitro CTWT (n = 7)Tg (n = 7)WT (n = 5)Tg (n = 5)BV/TV0.40 ± 0.020.25 ± 0.03**0.12 ± 0.010.04 ± 0.01**Tb.N (1/mm)3.19 ± 0.021.86 ± 0.25**3.86 ± 0.511.68 ± 0.34**Tb.Sp (mm)0.19 ± 0.010.48 ± 0.08**0.24 ± 0.030.68 ± 0.18*BMD (%)100% ± 15%52% ± 5%**100% ± 8%68% ± 6%** 14-week-old WT and Tg mice (n = 7 each group) were scanned using a small animal in vivo μCT scanner. Tibial bones were removed from 16-week-old WT and Tg mice (n = 5 each group) and scanned using an in vitro CT scanner. Analysis was performed on the proximal region of the tibia. The parameters calculated are Tb.N (trabecular number), Tb.Sp (trabecular separation), BV/TV (bone volume/tissue volume), and BMD (bone mineral density). Results shown are mean values ± SEM. BMD is given as relative to the average BMD of WT mice (**p < 0.01 vs WT mice). Low bone mass in gal-8 Tg mice is accompanied by enhanced bone remodeling To gain further insight into the mechanisms of reduced bone mass in the gal-8 Tg mice, bone formation parameters were determined by dynamic histomorphometry, using calcein doub" @default.
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W4233722201 title "Decision letter: The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice" @default.
W4233722201 doi "https://doi.org/10.7554/elife.05914.015" @default.
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