Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression.

Osteoblastic lineage cells (OBCs) are bone-building cells and essential component of hematopoietic niche, but mechanisms whereby bone-building and hematopoiesis-supportive activities of OBCs could be regulated simultaneously remain largely unknown. Here we found that B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) was involved in such a co-regulatory mechanism. In this study, we first found that, accompanied with marked decline of osteogenic activity, the hematopoietic niche in Bmi1 knockout (KO) mice was severely impaired and manifested as CXCL12 expression falls and LSK homing failure; however, intratibial injection with CXCL12 effectively facilitated LSK accumulation in bone marrow of Bmi1 KO mice. To try to rescue these defects in Bmi1 KO mice, we generated Bmi1KO/Sirt1Tg (KO-TG) double mutant mice with Sirt1 specific overexpression in mesenchymal progenitor cells (MPCs) in Bmi1 KO mice, and our data showed that KO-TG mice had significantly increased bone-building activity, elevated Cxcl12 expression by MPCs, increased LSK homing and expanded LSK pool in bone marrow compared to Bmi1 KO mice. Of note, similar improvements in KO-TG mice were observed in Bmi1 KO mice fed with dietary resveratrol, an established Sirt1 activator, comparing with KO control mice. Therefore, pharmacologic activation of Bmi1/Sirt1 signaling pathway could simultaneously promote bone-building and hematopoiesis-supportive activities of OBCs.

Overexpression of Sirt1 in mesenchymal stem cells protects against bone loss in mice by FOXO3a deacetylation and oxidative stress inhibition.

OBJECTIVE: B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) deficiency (Bmi-1-/-) leads to an osteoporotic phenotype with a significant downregulation of Sirt1 protein expression. Sirtuin 1 (Sirt1) haploinsufficiency results in a bone loss by decreased bone formation; however, it is unclear whether Sirt1 overexpression in mesenchymal stem cells (MSCs) plays an anti-osteoporotic role. The aim of the study is to identify whether the overexpression of Sirt1 in MSCs could restore skeletal growth retardation and osteoporosis in Bmi-1 deficient mice. METHODS: We used our new generated transgenic mouse model that overexpresses Sirt1 in its MSCs (Sirt1TG) to cross with Bmi-1-/- mice to generate Bmi-1-/- mice with Sirt1 overexpression in MSCs, and compared their skeletal metabolism with those of their Bmi-1-/- and wild-type (WT) littermates (6 mice for each genotype) at 4 weeks of age using imaging, histopathological, immunohistochemical, histomorphometric, cellular, and molecular methods. RESULTS: The levels of expression for Sirt1 were noticeably higher in the skeletal tissue of Sirt1TG mice than in those of WT mice. In Comparison to WT mice, the body weight and size, skeletal size, bone volume, osteoblast number, alkaline phosphatase and type I collagen positive areas, osteogenic related gene expression levels were all significantly increased in the Sirt1TG mice. Overexpression of Sirt1 in Bmi-1-/- mouse MSCs resulted in a longer lifespan, improved skeletal growth and significantly increased bone mass by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption in the Bmi-1-/- mice, although the defects were not completely restored. Furthermore, Sirt1 overexpression in MSCs reduced the acetylation level of FOXO3a (Forkhead box O3a), increasing levels of expression for FOXO3a and SOD2 (Superoxide dismutase 2) in bony tissue, enhanced osteogenesis and reduced osteogenic cell senescence. We also demonstrated that nicotinamide, a Sirt1 inhibitor, blocks the effect of overexpression of Sirt1 in MSCs on osteogenesis and osteogenic cell senescence. CONCLUSIONS: Taken together, these results demonstrate that Sirt1 overexpression in MSCs increased the osteoblastic bone formation and partially restores the defects in skeletal growth and osteogenesis in Bmi-1-/- mice by FOXO3a deacetylation and oxidative stress inhibition. Our data support the proposal that Sirt1 is a target for promoting bone formation as an anabolic approach for the treatment of osteoporosis.

Endocytosis of AtRGS1 Is Regulated by the Autophagy Pathway after D-Glucose Stimulation.

Sugar, as a signal molecule, has significant functions in signal transduction in which the seven-transmembrane regulator of G-protein signaling (RGS1) protein participates. D-Glucose causes endocytosis of the AtRGS1, leading to the physical uncoupling of AtRGS1 from AtGPA1 and thus a release of the GAP activity and concomitant sustained activation of G-protein signaling. Autophagy involves in massive degradation and recycling of cytoplasmic components to survive environmental stresses. The function of autophagy in AtRGS1 endocytosis during D-glucose stimulation has not been elucidated. In this study, we investigate the relationship between autophagy and AtRGS1 in response to D-glucose. Our findings demonstrated that AtRGS1 mediated the activation of autophagy by affecting the activities of the five functional groups of protein complexes and promoted the formation of autophagosomes under D-glucose application. When the autophagy pathway was interrupted, AtRGS1 recovery increased and endocytosis of ATRGS1 was inhibited, indicating that autophagy pathway plays an important role in regulating the endocytosis and recovery of AtRGS1 after D-glucose stimulation.

Bone marrow ablation demonstrates that estrogen plays an important role in osteogenesis and bone turnover via an antioxidative mechanism.

To assess the effect of estrogen deficiency on osteogenesis and bone turnover in vivo, 8-week-old mice were sham-operated or bilaterally ovariectomized (OVX), and after 8 weeks, mechanical bone marrow ablation (BMX) was performed and newly formed bone tissue was analyzed from 6 days to 2 weeks after BMX. Our results demonstrated that OVX mice following BMX displayed 2 reversed phase changes, one phase observed at 6 and 8 days after BMX delayed osteogenesis accompanied by a delay in osteoclastogenesis, and the other phase observed at 12 and 14 days after BMX increased osteoblastic activity and osteoclastic activity. Furthermore, we asked whether impaired osteogenesis caused by estrogen deficiency was associated with increased oxidative stress, and oxidative stress parameters were examined in bone tissue from sham-operated and OVX mice and OVX mice were administrated with antioxidant N-acetyl-l-cysteine (NAC) or vehicle after BMX. Results demonstrated that estrogen deficiency induced oxidative stress in mouse bone tissue with reduced antioxidase levels and activity, whereas NAC administration almost rescued the abnormalities in osteogenesis and bone turnover caused by OVX. Results from this study indicate that estrogen deficiency resulted in primarily impaired osteogenesis and subsequently accelerated bone turnover by increasing oxidative stress and oxidative stress promises to be an effective target in the process of treatment of postmenopausal osteoporosis.

[The role of Bmi-1 gene in submandibular gland of mice].

OBJECTIVE: To determine the role of Bmi-1 in the submandibular gland (SMG) of mice. METHODS: SMG of 4-week wild-type (WT) and Bmi-1 null (Bmi-1(-/-)) mice was analyzed on the weight, salivary flow rate, hematoxylin-eosin staining morphological differences and the changes in proliferation and aging by histology, immunohistochemistry and Western blotting. RESULTS: Compared with WT mice, the average static salivary flow rate [WT:(0.21 ± 0.02) µg/min,Bmi-1(-/-): (0.10 ± 0.02) µg/min] (P = 0.001) and the submandibular gland weight [WT: (1.89 ± 0.15) µg], Bmi-1(-/-): [(1.34 ± 0.07)µg] (P = 0.003) of the male Bmi-1(-/-) mice were significantly decreased, the number of gland duct increased, and the granular convoluted duct showed reduced diameter and branches. More senescence-associated ß-galactosidase positive cells existed in SMG of Bmi-1(-/-)mice (WT:0.00, Bmi-1(-/-): 0.18 ± 0.02), and Ki-67 immunopositive cells decreased in SMG of Bmi-1(-/-) mice (WT:0.40 ∼ 0.47, Bmi-1(-/-): 0.18 ∼ 0.20) (P = 0.000). The expression of p16 (WT:1.00 ± 0.12, Bmi-1(-/-): 0.00 ± 0.00) (P = 0.003) and p19 (WT:0.97 ± 0.09, Bmi-1(-/-): 5.09 ± 0.21) (P = 0.004) were up-regulated dramatically in SMG of the Bmi-1(-/-) mice. CONCLUSIONS: Bmi-1 gene deficiency causes abnormal function of SMG by inducing senescence phenotype of SMG.