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Zhao L.,Lanzhou University | Zhao L.,Orthopaedics Key Laboratory of Gansu Province | Zhao H.,Lanzhou University | Sheng X.,Lanzhou University | And 7 more authors.
Carbohydrate Polymers | Year: 2014

Radix Hedysari polysaccharides (HPS) is the principal active fraction of Radix Hedysari (RH). The information about HPS3d, the main fraction of HPS3, and its effect on bone is still unknown. In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide, 3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6 kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The FT-IR and elemental analysis showed that HPS3d was the sulfated polysaccharide. HPS3d upregulated alkaline phosphatase (ALP) activity and the expression of other osteogenic marker genes in osteoblast. In addition, HPS3d increased the expression and transcriptional activity of Runt-related transcription factor 2 (Runx-2) and Osterix, the two master genes of osteoblast differentiation. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of Runx-2 and Osterix. © 2014 Elsevier Ltd. Source

Jiang J.,Lanzhou University | Jiang J.,Orthopaedics Key Laboratory of Gansu Province | Zhao L.-G.,Lanzhou University | Zhao L.-G.,Orthopaedics Key Laboratory of Gansu Province | And 8 more authors.
Molecular and Cellular Biochemistry | Year: 2015

Bone cells respond to various mechanical stimuli including fluid shear stress (FSS) in vitro. Induction of cyclooxygenase-2 (COX-2) is thought to be important for the anabolic effects of mechanical loading. Recently, extracellular-signal-regulated kinase 5 (ERK5) has been found to be involved in multiple cellular processes. However, the relationship between ERK5 and the induction of COX-2 is still unknown. Here, we investigated the potential involvement of ERK5 in the response of pre-osteoblastic MC3T3-E1 cells upon FSS. MC3T3-E1 cells were subjected to 12 dyn/cm2 FSS. Then, we established a ERK5 small interfering RNA (siRNA) transfected cell line using the MC3T3-E1 cells. After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction and Western blotting, the expression of COX-2, cAMP response element-binding protein (CREB), and nuclear factor kappa B cells (NF-κB) were assayed for downstream effectors of activated ERK5 under FSS by Western blotting. Our results showed that FSS could stimulate COX-2 activity, and induce the phosphorylation of ERK5, CREB, and NF-κB. When the MC3T3-E1 cells were transfected using siRNA before exposure to FSS, COX-2 activity was suppressed, and the phosphorylation of CREB and NF-κB was significantly downregulated. In summary, we demonstrated that ERK5 pathway is essential in the induction of COX-2 gene. © 2015, Springer Science+Business Media New York. Source

Zhao L.-G.,Lanzhou University | Zhao L.-G.,Orthopaedics Key Laboratory of Gansu Province | Chen S.-L.,Lanzhou University | Chen S.-L.,Orthopaedics Key Laboratory of Gansu Province | And 7 more authors.
Connective Tissue Research | Year: 2014

The aim of this study was to determine the role of the mitogen-activated protein kinase kinase (MEK) 5/extracellular signal-regulated kinase (ERK) 5 pathway in osteoblast differentiation promoted by intermittent fluid shear stress (FSS). MC3T3-E1 osteoblastic cells were subjected to 12dyn/cm2 intermittent FSS, and the phenotypic markers for osteoblast differentiation, such as alkaline phosphatase (ALP) activity and expression of osteopontin (OPN) and osteocalcin (OCN), were then examined. The results showed that intermittent FSS could stimulate ERK5 phosphorylation, ALP activity and the expression of OPN and OCN. When the MEK5/ERK5 pathway was selectively inhibited by BIX02189, ALP activity was suppressed, and the expression of OPN and OCN was downregulated. Intermittent FSS induce the expression of Runt-related transcription factor-2 (Runx-2), which is involved in osteoblast differentiation by promoting the transcription of the above genes. Furthermore, the expression of Runx-2 was also reduced after treatment with BIX02189. Finally, we found that intermittent FSS was a more intense stimulus than steady FSS for promoting osteoblast differentiation. In summary, our results suggest that the MEK5/ERK5 pathway mediates osteoblast differentiation promoted by intermittent FSS, which was more effective than steady FSS in the differentiation process. The MEK5/ERK5 pathway also mediates FSS-induced Runx-2 expression in osteoblast differentiation. © 2014 Informa Healthcare USA, Inc. Source

Bin G.,Lanzhou University | Bin G.,Orthopaedics Key Laboratory of Gansu Province | Cuifang W.,Lanzhou University | Cuifang W.,Orthopaedics Key Laboratory of Gansu Province | And 16 more authors.
Biochemical and Biophysical Research Communications | Year: 2015

Fluid shear stress (FSS) is a potent mechanical stimulus and prevents cells from TNF-a-induced apoptosis. Recently, Extracellular-signal-regulated kinase 5 (ERK5) has been found to be involved in regulation of cell survival. However, little is known about the role of ERK5 signaling pathway in FSS-mediated anti-apoptotic effects in osteoblast. In this study, we show that FSS blocks TNF-a-induced apoptosis of MC3T3-E1 cells via ERK5 signaling pathway. We found that physiological FSS for 1 h significantly decreased TNF-α-induced MC3T3-E1 cells apoptosis. After inhibition of ERK5 activity by XMD8-92, a highly-selective inhibitor of ERK5 activity, the ability of FSS to inhibit TNF-α induced apoptosis was significantly decreased. Analysis of anti-apoptotic mechanisms indicated that exposure of MC3T3-E1 cells to FSS for 1 h increased phosphorylation of Bad and inhibited caspase-3 activity. After treatment with XMD8-92, phosphorylation of Bad by FSS was significantly blocked, but caspase-3 activity was increased. In summary, these findings indicated that FSS inhibits TNF-α-mediated signaling events in osteoblast by a mechanism dependent on activation of ERK5, and Bad is a crucial downstream target for ERK5. Those results implied that ERK5 signaling pathway play a crucial role in FSS-mediated anti-apoptotic effect in osteoblast. Thus, ERK5 signaling pathway may be a new drug treatment target of osteoporosis and related bone-wasting diseases. © 2015 Elsevier Inc. All rights reserved. Source

Bin G.,Lanzhou University | Bin G.,Orthopaedics Key Laboratory of Gansu Province | Bo Z.,Lanzhou University | Bo Z.,Orthopaedics Key Laboratory of Gansu Province | And 18 more authors.
Experimental Cell Research | Year: 2016

TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. © 2016 Elsevier Inc. Source

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