Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The inhibition of PKM2 activity by IgSF11 is required for osteoclast differentiation. a Effect of shikonin on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of shikonin was added and incubated for three days. b Effect of TEPP46 on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of TEPP46 was added and incubated for three days. TRAP staining and the frequency of TRAP + multinucleated cells (3 nuclei or more per cell) are shown. Scale bars represent 100 μm. c Lactate production during osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The lactate concentration in the culture supernatants was measured daily. d Lactate production in response to the modulation of PKM2 activity. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. Control DMSO or TEPP46 was added to IgSF11 +/+ cultures and incubated for three days. Control DMSO or shikonin was added to IgSF11 −/− cultures and incubated for three days. The lactate concentrations in the culture supernatants were measured. e Effect of glycolysis intermediates on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of glycolysis intermediates was added to IgSF11 −/− cultures and incubated for three days. IgSF11 +/+ cultures were used as a control. The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown. The data are presented as the means ± S.D. Each dot represents a technical replicate. The results are representative of at least three independent experiments

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Inhibition, Activity Assay, Cell Culture, Incubation, Staining, Concentration Assay

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: Proper PKM2 activity is required for bone homeostasis. a , d Representative microcomputed tomography (μCT) images of femurs from mice treated with shikonin ( n = 5 mice per group) for 4 weeks ( a ) or TEPP46 ( n = 5 mice per group) for 6 weeks ( d ). Bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), bone mineral density (BMD), and cortical thickness (Ct.Th) are shown. Scale bars represent 0.5 mm. b , e Representative TRAP staining images of the tibias of mice treated with shikonin for 4 weeks ( b ) or TEPP46 for 6 weeks ( e ). The scale bar represents 200 μm. GP Growth plate, Metap. Metaphysis. c , f Representative dynamic histomorphometry of the tibias of mice treated with shikonin for 4 weeks ( c ) or TEPP46 for 6 weeks ( f ). The mineral apposition rate (MAR) and bone formation rate (BFR) are shown. The scale bar represents 50 μm. Each dot represents the result of a single mouse ( n = 5 mice per group). Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activity Assay, Tomography, Staining

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The activation of PKM2 attenuates LPS-induced osteoclastic bone loss. a Representative μCT images of mouse calvaria 5 days after LPS injection with or without TEPP46 ( n = 5–6 mice per group). The scale bar represents 1 mm. Percentages of the bone destruction areas (Des. area) in calvaria are shown. b Representative TRAP staining images of calvarial coronal sections in ( a ). TRAP + osteoclast size (OC size) and number (OC.N/BS) are shown. Scale bar represents 200 μm. c Serum levels of TNF-α, IL-6, and IL-17AF in the mice in ( a ) are shown. d Representative μCT images of mouse calvaria at 4 days after RANKL injection with or without TEPP46 are shown ( n = 4–6 mice per group). The scale bar represents 1 mm. e Representative TRAP staining images of calvarial coronal sections in ( d ). The scale bar represents 200 μm. Each dot represents the result of a single mouse. Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activation Assay, Injection, Staining

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The activation of PKM2 mitigates colitis-induced osteoclastic bone loss. a Representative μCT images of the femurs of mice treated with DSS or the control with or without TEPP46. Bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), bone mineral density (BMD), and cortical thickness (Ct.Th) are shown ( n = 4–6 mice per group). Scale bars represent 0.5 mm. b Representative TRAP staining images of tibias in ( a ). TRAP + osteoclast size (OC size) and number (OC.N/BS) are shown. The scale bar represents 200 μm. GP Growth plate, Metap. Metaphysis. c Representative dynamic histomorphometry of tibias in Panel ( a ). The mineral apposition rate (MAR) and bone formation rate (BFR) are shown. The scale bar represents 20 μm. d Serum levels of TNF-α, IL-6, and IL-17AF from the mice in ( a ) were measured by ELISA. Each dot represents the result of a single mouse. Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: Schematic depicting the role of IgSF11-mediated regulation of PKM2 activity during osteoclast differentiation. a PKM2 activity is high in BMMs. RANKL stimulation enhances the expression of IgSF11. b IgSF11 mediates the phosphorylation of PKM2 through Src family kinases (c-Src, Fyn, and HcK) in a manner dependent on the C-terminal region. This phosphorylation leads to the inhibition of PKM2 activity. In the absence of IgSF11, the inhibition of PKM2 activity barely occurs, keeping PKM2 activity high. c IgSF11-mediated inhibition of PKM2 activity increases osteoclast differentiation. In the absence of IgSF11, osteoclast differentiation is impaired due to a lack of PKM2 inhibition, which can be rescued by the PKM2-specific inhibitor shikonin. Moreover, the PKM2-specific activator TEPP46 inhibits osteoclast differentiation in the presence of IgSF11

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activity Assay, Expressing, Inhibition