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/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.

Kindlin-2 mediates mechanotransduction in bone by regulating expression of Sclerostin in osteocytes

“…Fluid-induced flow shear stress was performed with Streamer ® System STR-4000 (Flexcell International Corporation, Burlington, NC, USA) as discussed previously in Chen et al 92 . MLO-Y4 cells with a total cell number of 3.0 × 10 5 cells were seed on Collagen-I pre-coated culture slips (75 mm × 25 mm × 1 mm, Flexcell) for 48 h before FSS treatment.…”

Chromium Oxide Nanoparticle Impaired Osteogenesis and Cellular Response to Mechanical Stimulus

“…Oscillatory fluid flow was achieved by using the Flexcell Streamer parallel plate flow chamber system Flexcell Streamer System (FX4000, Flexcell International, USA) as illustrated. 24 The apparatus was placed in an incubator for the duration of the experimental maintained at 37°C under 5% CO 2 . The cells were seeded on the type I collagen-coated glass slides (Flexcell, USA) for 24 h prior to experiment.…”

Hydroxysafflor yellow A attenuates oxidative stress injury‑induced apoptosis in the nucleus pulposus cell line and regulates extracellular matrix balance via CA XII

“…NP cell identification. The primary NP cells were cultured in 6-well plates, at a density of 1x10 5 , and a coverslip was placed in the bottom layer for cell climbing. After 24 h, the coverslips were removed and the NP cells were washed with cold PBS, fixed in 4% paraformaldehyde at 4˚C for 15 min, permeabilized with 0.5% Triton X-100 for 20 min, then blocked with 1% BSA (Proliant Biologicals, LLC) for 30 min at room temperature.…”

“…It is well-known that nucleus pulposus (NP) dysfunction has been associated with IVDD. In NP tissues, NP cells play an essential role in maintaining extracellular matrix (ECM) homeostasis (4)(5)(6). It has been demonstrated that excessive apoptosis of the NP cells, caused by oxidative stress, inflammation and mechanical load, which subsequently leads to exacerbated ECM degradation, could be a critical pathogenic mechanism of disc degeneration (7).…”