Dataset Information

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

ABSTRACT: 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/cm2) 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/cm2) 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.

SUBMITTER: Chen S

PROVIDER: S-EPMC7064043 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

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

Chen Sheng S Qin Lei L Wu Xiaohao X Fu Xuekun X Lin Sixiong S Chen Di D Xiao Guozhi G Shao Zengwu Z Cao Huiling H

Frontiers in cell and developmental biology 20200303

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<sup>2</sup>) increases the sulfated glycosaminoglycan (sGAG) content and protein levels of Col2a1 and Aggrecan and d ...[more]

PMID: 32195253

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Project description:Intervertebral disc degeneration (IDD) is the primary pathological mechanism that underlies low back pain. Overloading-induced cell death, especially endogenous stem cell death, is the leading factor that undermines intrinsic repair and aggravates IDD. Previous research has separately studied the effect of oxygen concentration and mechanical loading in IDD. However, how these two factors synergistically influence endogenous repair remains unclear. Therefore, we established in vitro and in vivo models to study the mechanisms by which hypoxia interacted with overloading-induced cell death of the nucleus pulposus derived stem cells (NPSCs). We found the content of HIF1A (hypoxia inducible factor 1 subunit alpha) and the number of NPSCs decreased with disc degeneration in both rats and human discs. Hence, we isolated this subpopulation from rat discs and treated them simultaneously with hypoxia and excessive mechanical stress. Our results demonstrated that hypoxia exerted protective effect on NPSCs under compression, partially through elevating macroautophagy/autophagy. Proteomics and knockdown experiments further revealed HIF1A-BNIP3-ATG7 axis mediated the increase in autophagy flux, in which HMOX1 and SLC2A1 were also involved. Moreover, HIF1A-overexpressing NPSCs exhibited stronger resistance to over-loading induced apoptosis in vitro. They also showed higher survival rates, along with elevated autophagy after being intra-disc transplanted into over-loaded discs. Jointly, both in vivo and in vitro experiments proved the anti-apoptotic effect of HIF1A on NPSCs under the excessive mechanical loading, suggesting that restoring hypoxia and manipulating autophagy is crucial to maintain the intrinsic repair and to retard disc degeneration.Abbreviations: 3-MA: 3-methyladenine; ACAN: aggrecan; ATG7: autophagy related 7; BafA1: bafilomycin A1; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CCK8: cell counting kit-8; CHT: chetomin; CMP: compression; CoCl2: cobalt chloride; COL2A1: collagen type II alpha 1 chain; Ctrl: control; DAPI: 4,6-diamidino-2-phenylindole; DEP: differentially expressed protein; DiR: 1,1-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine; ECM: extracellular matrix; FCM: flow cytometry; GD2: disialoganglioside GD 2; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; H&E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HK2: hexokinase 2; HMOX1: heme oxygenase 1; HX: hypoxia mimicry; IDD: intervertebral disc degeneration; IF: immunofluorescence; IHC: immunohistochemistry; IVD: intervertebral disc; KEGG: kyoto encyclopedia of genes and genomes; LBP: low back pain; Lv: lentivirus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NC: negative control; NIR: near-infrared; NP: nucleus pulposus; NPC: nucleus pulposus cell; NPSC: nucleus pulposus derived stem cell; NX: normoxia; PPI: protein-protein interactions; RFP: red fluorescent protein; SLC2A1/GLUT1: solute carrier family 2 member 1; SQSTM1/p62: sequestosome 1; TEK/TIE2: TEK receptor tyrosine kinase; TEM: transmission electron microscopy; TUBB: tubulin beta class I.

Dataset Information

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

Publications

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

Similar Datasets

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