Fluid Shear Stress Suppresses Osteoclast Differentiation in RAW264.7 Cells through Extracellular Signal-Regulated Kinase 5 (ERK5) Signaling Pathway

Abstract

Background:Although extracellular signal-regulated kinase 5 (ERK5) is known to be critical for osteoclast differentiation, there are few studies on how fluid shear stress (FSS) regulates osteoclast differentiation through the ERK5 signaling pathway. We examined the expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and its downstream factors, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9) and their relationship with ERK5. Material/Methods: RAW264.7 cells were treated with RANKL, XMD8-92 (ERK5 inhibitor), and then loaded onto 12 dyn/cm 2 FSS for 4 days. Endpoints measured were osteoclast differentiation, bone resorption, and TRAP activity. Cell viability was detected by using the Cell Counting Kit-8 (CCK-8) assay. Western blot was used to analyze protein expression of phosphorylated-ERK5 (p-ERK5), NFATc1, CTSK, TRAP, and MMP-9. Results:FSS inhibited osteoclast differentiation and expression of NFATc1, CTSK, TRAP, and MMP-9; cell viability was not affected. ERK5 expression increased by FSS but not by RANKL, and it was blocked by XMD8-92. Furthermore, FSS suppressed osteoclast differentiation in RAW264.7 cells through ERK5 pathway. Conclusions:Our findings demonstrated that FSS inhibited osteoclast differentiation in RAW264.7 cells via the ERK5 pathway through reduced NFATc1 expression and its downstream factors MMP-9, CTSK, and TRAP.

Calcitriol inhibits osteoclastogenesis in an inflammatory environment by changing the proportion and function of T helper cell subsets (Th2/Th17)

“…Furthermore, the levels of osteoclastogenesis‐related genes and proteins (such as NFATc1 and MMP‐9) were determined by qRT‐PCR and Western blot analysis (for details, see Sections 2.8 and 2.9). 33 …”

“…In comparison to incubation without calcitriol, incubation with calcitriol led to fewer RAW264.7 cells exhibiting positive TRAP staining and RAW264.7 cells with lower levels of NFATc1 and MMP-9 (Figure3A-D), which suggests that Th cell polarization is a crucial part of the process of the calcitriol-mediated suppression of osteoclastogenesis. NFATc1 and MMP-9 were selected as osteoclastogenesis-related markers in this research 33. NFATc1, as an essential transcription factor in osteoclasts, can regulate cathepsin K and TRAP during bone resorption and be inhibited by low levels of RANKL due to low Th17 and high Th2 responses 8,45.…”

Mechanical stimulation controls osteoclast function through the regulation of Ca2+-activated Cl− channel Anoctamin 1

“…The correlation between FSS and flow rate was calculated using the equation: τ = 6μQ/bh2, where Q is the flow rate (cm 3 / s), μ is the viscosity of the flow media (0.01 dynes/cm2), h is the height of the channel (0.05 cm), b is the slit width (2.5 cm), and τ is the wall shear stress (dyne/ cm 2 ). The parallel plate flow chamber has been described previously 8,46 .…”

Mechanical regulation of bone remodeling

“…FSS has been reported to affect the cell morphology and gene expression of osteoclasts and to inhibit their differentiation without affecting their viability. 22 – 24 FSS also stimulates nitric oxide (NO) and prostaglandin E 2 (PGE2) production in bone marrow-derived preosteoclast-like cells. 25 Studies of ion channels also provide evidence of the mechanosensitive capabilities of osteoclasts.…”