Yaron Vinik

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice

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. DOI: http://dx.doi.org/10.7554/eLife.05914.001

Otubain 2 is a novel promoter of beta cell survival as revealed by siRNA high-throughput screens of human pancreatic islets

Aims/hypothesisPro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches.MethodsSmall interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1β and IFN-γ were transfected with ∼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified.ResultsDispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment.ConclusionsUse of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.

Prolonged insulin treatment sensitizes apoptosis pathways in pancreatic β cells.

Insulin resistance results from impaired insulin signaling in target tissues that leads to increased levels of insulin required to control plasma glucose levels. The cycle of hyperglycemia and hyperinsulinemia eventually leads to pancreatic cell deterioration and death by a mechanism that is yet unclear. Insulin induces ROS formation in several cell types. Furthermore, death of pancreatic cells induced by oxidative stress could be potentiated by insulin. Here, we investigated the mechanism underlying this phenomenon. Experiments were done on pancreatic cell lines (Min-6, RINm, INS-1), isolated mouse and human islets, and on cell lines derived from nonpancreatic sources. Insulin (100nM) for 24h selectively increased the production of ROS in pancreatic cells and isolated pancreatic islets, but only slightly affected the expression of antioxidant enzymes. This was accompanied by a time- and dose-dependent decrease in cellular reducing power of pancreatic cells induced by insulin and altered expression of several ER stress response elements including a significant increase in Trb3 and a slight increase in iNos The effect on iNos did not increase NO levels. Insulin also potentiated the decrease in cellular reducing power induced by H2O2 but not cytokines. Insulin decreased the expression of MCL-1, an antiapoptotic protein of the BCL family, and induced a modest yet significant increase in caspase 3/7 activity. In accord with these findings, inhibition of caspase activity eliminated the ability of insulin to increase cell death. We conclude that prolonged elevated levels of insulin may prime apoptosis and cell death-inducing mechanisms as a result of oxidative stress in pancreatic cells.

Ablation of the mammalian lectin galectin-8 induces bone defects in mice

Mice overexpressing galectin‐8 [gal‐8 transgenic (Tg)], a secreted mammalian lectin, exhibit enhanced bone turnover and reduced bone mass, similar to cases of postmenopausal osteoporosis. Here, we show that gal‐8 knockout (KO) mice have increased bone mass accrual at a young age but exhibit accelerated bone loss during adulthood. These phenotypes can be attributed to a gal‐8‐mediated increase in receptor activator of NF‐κB ligand (RANKL) expression that promotes osteoclastogenesis, combined with direct inhibition of osteoblast differentiation, evident by reduced bone morphogenetic protein (BMP) signaling, reduced phosphorylation of receptor regulated mothers against decapentaplegic homolog (R‐SMAD) and reduced expression of osteoblast differentiation markers osterix, osteocalcin, runt‐related transcription factor 2 (RUNX2), dentin matrix acidic phosphoprotein‐1 (DMP1), and alkaline phosphatase. At the same time, gal‐8 promotes expression of estrogen receptor a (ESR1). Accordingly, the rate of bone loss is accelerated in ovariectomized, estrogen‐deficient gal‐8 Tg mice, whereas gal‐8 KO mice, having low levels of ESR1, are refractory to ovariectomy. Finally, gal‐8 mRNA positively correlates with the mRNA levels of osteoclastogenic markers RANKL, tartrate‐resistant acid phosphatase, and cathepsin K in human femurs. Collectively, these findings identify gal‐8 as a new physiologic player in the regulation of bone mass.—Vinik, Y., Shatz‐Azoulay, H., Hiram‐Bab, S., Kandel, L., Gabet, Y., Rivkin, G., Zick, Y. Ablation of the mammalian lectin galectin‐8 induces bone defects in mice. FASEB J. 32, 2366–2380 (2018). www.fasebj.org

Prolonged Elimination of Negative Feedback Control Mechanisms Along the Insulin Signalling Pathway Impairs β Cell Function In Vivo

Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-I signaling. We therefore examined the effects of mutation of five “inhibitory” Ser phosphorylation sites on IRS2 function in transgenic mice that overexpress, selectively in pancreatic β-cells, either wild-type (WT) or a mutated IRS2 protein (IRS25A). Islets size, number, and mRNA levels of catalase and superoxide dismutase were increased, whereas those of nitric oxide synthase were decreased, in 7- to 10-week-old IRS25A-β mice compared with IRS2WT-β mice. However, glucose homeostasis and insulin secretion in IRS25A-β mice were impaired when compared with IRS2WT-β mice or to nontransgenic mice. This was associated with reduced mRNA levels of Glut2 and islet β-cell transcription factors such as Nkx6.1 and MafA. Similarly, components mediating the unfolded protein response were decreased in islets of IRS25A-β mice in accordance with their decreased insulin secretion. The beneficial effects of IRS25A on β-cell proliferation and β-cell transcription factors were evident only in 5- to 8-day-old mice. These findings suggest that elimination of inhibitory Ser phosphorylation sites of IRS2 exerts short-term beneficial effects in vivo; however, their sustained elimination leads to impaired β-cell function.

Corrigendum: Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8

The Table of Contents (ToC) graphic and Figure 2 are incorrect in this article as it first appeared online on June 21, 2018 and in Volume 13, Issue 17, pages 1664–1672. The error pertains to an incorrect depiction of the carbohydrate 3′‐sialyllactose, specifically the glucose moiety of the lactose portion of 3′‐sialyllactose. The correct Table of Contents graphic / Figure 2 is shown here (same graphic used for both). The correction has no bearing on the conclusions presented in the article.

Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8

Galectin‐8 is a β‐galactoside‐recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3‐O‐[1‐carboxyethyl]‐β‐d‐galactopyranoside (compound 6) as a ligand to target the N‐terminal domain of galectin‐8 (galectin‐8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3′‐O‐sialylated lactose (3′‐SiaLac), with galectin‐8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin‐8N for 6 was 32.8 μm, whereas no significant affinity was detected for the C‐terminal domain of galectin‐8 (galectin‐8C). The crystal structure of the galectin‐8N–6 complex validated the predicted binding conformation and revealed the exact protein–ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin‐8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide‐based scaffold as a binder of galectin‐8.