Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytes in vitro

Zhang, Bingbing; Hou, Rutao; Zou, Zhen; Luo, Tao; Zhang, Yang; Wang, Liyun; Wang, Bin

doi:10.1016/j.redox.2017.10.021

Cited by 61 publications

(47 citation statements)

References 35 publications

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“…We have also tested the immediate response of OCY454 osteocytes to flow, in an attempt to verify and quantify its mechano‐sensitivity. Flow‐induced ATP release is a well‐known indicator of mechanotransduction for osteocytes, often coupled with autophagy and transient calcium response . Similar to the results observed for MLO‐Y4 cells, the new OCY454 osteocytes also exhibited a significant increase in total ATP expression when stimulated with flow.…”

Section: Discussionsupporting

confidence: 71%

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OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

Shao

Vivian

et al. 2019

Journal Orthopaedic Research

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Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies.

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Section: Discussionsupporting

confidence: 71%

“…The cells in the devices were then stimulated with OFF (2 Pa, 1 Hz) for 20 min. Cell lysate and collected media were combined for the quantification of total ATP synthesis, as mentioned previously . ATP levels were measured using a luciferin‐luciferase reaction (Abcam, Cambridge, UK).…”

Section: Methodsmentioning

confidence: 99%

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

Shao

Vivian

et al. 2019

Journal Orthopaedic Research

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“…For MDA and SOD measurement (Y. Zhang, Zou, et al, 2018), the samples were rinsed with PBS and then homogenized and sonicated in lysis buffer on ice (B. Zhang, Hou, et al, 2018).…”

Section: Measurement Of Malondialdehyde (Mda) Glutathione (Gsh) Amentioning

confidence: 99%

“…Zhang, Zou, et al, 2018), the samples were rinsed with PBS and then homogenized and sonicated in lysis buffer on ice (B. Zhang, Hou, et al, 2018). After sonication, the lysed tissues were centrifuged at 10,000g for 10 min to remove debris (Riehle & Bauersachs, 2018).…”

Section: Measurement Of Malondialdehyde (Mda) Glutathione (Gsh) Amentioning

confidence: 99%

PTEN inhibition attenuates endothelial cell apoptosis in coronary heart disease via modulating the AMPK–CREB–Mfn2‐mitophagy signaling pathway

Wang

Zhao

et al. 2019

Journal Cellular Physiology

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Atherosclerosis (AS) is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in the occurrence and development of AS. In our study, we attempted to evaluate the role of phosphatase and tensin homolog (PTEN) in endothelial cell apoptosis under oxidized low-density lipoprotein (ox-LDL) stimulation and identify the associated mechanisms. The results of our study demonstrated that ox-LDL induced human umbilical vein endothelial cell (HUVEC) death via mitochondrial apoptosis, as evidenced by reduced mitochondrial potential, increased mitochondria permeability transition pore opening, cellular calcium overload, and caspase-9/-3 activation. In addition, ox-LDL also suppressed cellular energy production via downregulating the mitochondrial respiratory complex. Moreover, ox-LDL impaired HUVECs migration. Western blot analysis showed that PTEN expression was upregulated after exposure to ox-LDL and knockdown of PTEN could attenuate ox-LDL-mediated endothelial cell damage. Furthermore, we found that ox-LDL impaired mitophagy activity, whereas PTEN deletion could improve mitophagic flux and this effect relied on the activity of the AMPactivated protein kinase (AMPK)-cAMP-response element-binding protein (CREB)-Mitofusin-2 (Mfn2) axis. When the AMPK-CREB-Mfn2 pathway was inhibited, PTEN deletion-associated HUVECs protection was significantly reduced, suggesting that the AMPK-CREB-Mfn2-mitophagy axis is required for PTEN deletion-mediated endothelial cell survival under ox-LDL. Taken together, our results indicate that ox-LDL-induced endothelial cell damage is associated with PTEN overexpression, and inhibition of PTEN could promote endothelial survival via activating the AMPK-CREB-Mfn2-mitophagy signaling pathway. K E Y W O R D S AMPK-CREB-Mfn2 signaling pathway, endothelial cell dysfunction, Mfn2, mitophagy, ox-LDL, PTEN

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“…Fluid shear stress activates autophagy in multiple cell types (Zhang et al, 2018;Yang et al, 2019). Thus, we determined whether FSS activates autophagy in NP cells.…”

Section: Fss Promotes Autophagy In Np Cellsmentioning

confidence: 99%

Moderate Fluid Shear Stress Regulates Heme Oxygenase-1 Expression to Promote Autophagy and ECM Homeostasis in the Nucleus Pulposus Cells

Chen

Qin

et al. 2020

Front. Cell Dev. Biol.

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In vertebrate, the nucleus pulposus (NP), which is an essential component of the intervertebral disk, is constantly impacted by fluid shear stress (FSS); however, molecular mechanism(s) through which FSS modulates the NP homeostasis is poorly understood. Here we show that FSS regulates the extracellular matrix (ECM) homeostasis in NP cells. A moderate dose of FSS (i.e., 12 dyne/cm 2) increases the sulfated glycosaminoglycan (sGAG) content and protein levels of Col2a1 and Aggrecan and decreases those of matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADMATS5) in rat NP cells, while a higher dose of FSS (i.e., 24 dyne/cm 2) displays opposite effects. Results from RNA sequencing analysis, quantitative real-time RT-PCR analysis and western blotting establish that the heme oxygenase-1 (HO-1) is a key downstream mediator of the FSS actions in NP cells. HO-1 knockdown abolishes FSS-induced alterations in ECM protein production and sGAG content in NP cells, which is reversed by HO-1 induction. Furthermore, FSS activates the autophagic pathway by increasing the LC3-II/LC3-I ratio, Beclin-1 protein level, and formation of autophagosome and autolysosome and thereby regulates ECM protein and sGAG production in a HO-1 dependent manner. Finally, we demonstrate that the intraflagellar transport (IFT) 88, a core trafficking protein of primary cilia, is critically involved in the HO-1-mediated autophagy activation and ECM protein and sGAG production in FSS-treated NP cells. Thus, we for the first time demonstrate that FSS plays an important role in maintaining ECM homeostasis through HO-1-dependent activation of autophagy in NP cells.

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Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytes in vitro

Cited by 61 publications

References 35 publications

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

PTEN inhibition attenuates endothelial cell apoptosis in coronary heart disease via modulating the AMPK–CREB–Mfn2‐mitophagy signaling pathway

Moderate Fluid Shear Stress Regulates Heme Oxygenase-1 Expression to Promote Autophagy and ECM Homeostasis in the Nucleus Pulposus Cells

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