Xiaochun Bai

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-κB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Anti‐diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose‐dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3‐E1. Inhibition of AMP‐activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin‐induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)‐positive multi‐nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP‐positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats. J. Cell. Biochem. 112: 2902–2909, 2011.

miR-483-5p Promotes Invasion and Metastasis of Lung Adenocarcinoma by Targeting RhoGDI1 and ALCAM

The nodal regulatory properties of microRNAs (miRNA) in metastatic cancer may offer new targets for therapeutic control. Here, we report that upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. miR-483-5p promotes the epithelial-mesenchymal transition (EMT) accompanied by invasive and metastatic properties of lung adenocarcinoma. Mechanistically, miR-483-5p is activated by the WNT/β-catenin signaling pathway and exerts its prometastatic function by directly targeting the Rho GDP dissociation inhibitor alpha (RhoGDI1) and activated leukocyte cell adhesion molecule (ALCAM), two putative metastasis suppressors. Furthermore, we found that downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. Importantly, miR-483-5p levels are positively correlated with β-catenin expression, but are negatively correlated with the levels of RhoGDI1 and ALCAM in human lung adenocarcinoma. Our findings reveal that miR-483-5p is a critical β-catenin-activated prometastatic miRNA and a negative regulator of the metastasis suppressors RhoGDI1 and ALCAM.

Metformin inhibits renal cell carcinoma in vitro and in vivo xenograft

OBJECTIVE To evaluate the effects of metformin on renal cell carcinoma (RCC) and its underlying mechanisms. MATERIALS AND METHODS We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assays to investigate the effects of metformin on RCC cell growth. Flow cytometry was used to evaluate the cell cycle changes after metformin treatment. We further determined the possible signaling molecules involved in this process by immunoblot analysis of various proteins. Furthermore, a xenograft model was used to study the effects of metformin on RCC tumor growth. RESULTS We demonstrated that metformin effectively inhibits cell proliferation in 786-O and OS-RC-2 RCC cell lines. Moreover, metformin down-regulated cyclin D1 expression and induced G0/G1 cell cycle arrest in these cells. Further study revealed metformin induced the activation of AMP-activated protein kinase (AMPK), and inhibited mammalian target of rapamycin (mTOR), which is a central regulator of protein synthesis and cell growth, and negatively regulated by AMPK. Most importantly, daily treatment of mice with metformin prevented RCC tumor growth in a xenograft model. CONCLUSIONS Metformin was able to induce G0/G1 cell cycle arrest and inhibit RCC growth in vitro and in vivo. These results suggest that metformin may be a potential therapeutic agent for the treatment of RCC.

Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell Proliferation in Osteoblasts

Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5–200 μM hydrogen peroxide (H2O2) dose‐ and time‐dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3‐E1, and in human osteoblast‐like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G2/M phase increased about 2‐4 fold by 200 μM H2O2 treatment for 24–72 hr. A total of 0.5–5 mM of H2O2 but not lower concentrations (5–200 μM) of H2O2 inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E‐BP1 (T37/46), and S6(S235/236) in MC3T3‐E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H2O2 prevents cell proliferation of osteoblasts by down‐regulating cyclin B1 and inducing G2 cell cycle arrest. Inhibition of mTOR signaling by H2O2 may not be involved in this process. Anat Rec, 292:1107–1113, 2009.

Targeting of mTORC2 prevents cell migration and promotes apoptosis in breast cancer

Most of breast cancers are resistant to mammalian target of rapamycin complex 1 (mTORC1) inhibitors rapamycin and rapalogs. Recent studies indicate mTORC2 is emerging as a promising cancer therapeutic target. In this study, we compared the inhibitory effects of targeting mTORC1 with mTORC2 on a variety of breast cancer cell lines and xenograft. We demonstrated that inhibition of mTORC1/2 by mTOR kinase inhibitors PP242 and OSI-027 effectively suppress phosphorylation of Akt (S473) and breast cancer cell proliferation. Targeting of mTORC2 either by kinase inhibitors or rictor knockdown, but not inhibition of mTORC1 either by rapamycin or raptor knockdown promotes serum starvation- or cisplatin-induced apoptosis. Furthermore, targeting of mTORC2 but not mTORC1 efficiently prevent breast cancer cell migration. Most importantly, in vivo administration of PP242 but not rapamycin as single agent effectively prevents breast tumor growth and induces apoptosis in xenograft. Our data suggest that agents that inhibit mTORC2 may have advantages over selective mTORC1 inhibitors in the treatment of breast cancers. Given that mTOR kinase inhibitors are in clinical trials, this study provides a strong rationale for testing the use of mTOR kinase inhibitors or combination of mTOR kinase inhibitors and cisplatin in the clinic.

The switch I region of Rheb is critical for its interaction with FKBP38.

The Ras-like small GTPase Rheb is an upstream activator of the mammalian target of rapamycin (mTOR). It has recently been shown that Rheb activates mTOR by binding to its endogenous inhibitor FKBP38 and preventing it from association with mTOR. The interaction of Rheb with FKBP38 is controlled by its guanine nucleotide binding states, which are responsive to growth factor and amino acid conditions. In this study, we show that Rheb interacts with FKBP38 through a section within its switch I region that is equivalent to the effector domain of other Ras-like small GTPases. We find that the ability for Rheb to interact with FKBP38 correlates with its activity for mTOR activation. Our findings suggest that FKBP38 is a bona fide effector of Rheb and that the ability to interact with FKBP38 is important for Rheb as an activator of mTOR.

Click chemistry plays a dual role in biodegradable polymer design

Click chemistry plays a dual role in the design of new citrate-based biodegradable elastomers (CABEs) with greatly improved mechanical strength and easily clickable surfaces for biofunctionalization. This novel chemistry modification strategy is applicable to a number of different types of polymers for improved mechanical properties and biofunctionality.

Inhibition of mTOR signaling by oleanolic acid contributes to its anti-tumor activity in osteosarcoma cells†

Oleanolic acid (OA), a pentacyclic triterpenoid exhibits potent anti‐tumor activity against many tumor cell lines. But the mechanisms through which OA inhibits osteosarcoma cells are not known. The mammalian target of rapamycin (mTOR) serves as a central regulator of cell growth, proliferation, survival, and metabolism by integrating intracellular and extracellular signals. In this study, we examined effects of OA on proliferation, cell cycle progression, apoptosis in osteosarcoma cells, and involvement of mTOR signaling in this process. OA inhibited cell proliferation and colony formation, induced G1 arrest in osteosarcoma MG63 and Saos‐2 cells dose and time dependently. The protein level of cyclin D1, which plays critical role in G1 to S phase transition and servers as a downstream target of mTOR complex 1 (mTORC1) was down‐regulated by OA. Phosphorylation of p70 ribosomal S6 kinase 1 (p70 S6K1) (T389) and S6 (S235/236), mediators of mTORC1 signaling in controlling protein translation and cell growth, was also inhibited by OA. Furthermore, OA inhibited phosphorylation of Akt, a pro‐survival factor and substrate for mTORC2. Inactivation of Akt correlated with pro‐apoptotic role of OA in osteosarcoma cells, as manifested by an increase in annexin V‐FITC binding, cleavage of poly (ADP‐ribose) polymerase (PARP) and activation of caspases 3. Our results suggest that OA is a promising agent for treatment of osteosarcoma and mTOR signaling may contribute to its anti‐tumor effects on osteosarcoma cells.

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known.We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type dependent. In HEK293 cells hydrogen peroxide (H(2)O(2)) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H(2)O(2)-induced Raptor (S792) phosphorylation and reverses H(2)O(2)-induced dephosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H(2)O(2) also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H(2)O(2). Moreover, inhibition of PP2A and AMPK partially rescued cells from H(2)O(2)-induced cell death. High doses of H(2)O(2) inhibit while low doses of H(2)O(2) activate mTORC1 both in TSC2(-/-) P53(-/-) and TSC2(+/+) P53(-/-) MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H(2)O(2)-induced inhibition of mTORC1 signaling.