METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling.

Yiwen Zhang,Qiong Xu,Xiaofei Gu,Di Li,Luhui Cai

doi:10.3390/ijms21010199

Yiwen Zhang, Qiong Xu + Show 3 more

Open Access

https://doi.org/10.3390/ijms21010199

Copy DOI

Abstract

Osteoblasts are crucial bone-building cells that maintain bone homeostasis, whereas inflammatory stimuli can inhibit osteogenesis and activate inflammatory response. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes and plays important roles in multiple biological processes. However, whether m6A modification affects osteoblast differentiation and inflammatory response remains unknown. To address this issue, we investigated the expression of the N6-adenosine methyltransferase METTL3 and found that it was upregulated during osteoblast differentiation and downregulated after LPS stimulation. We then knocked down METTL3 and observed decreased levels of osteogenic markers, ALP activity, and mineralized nodules, as well as Smad1/5/9 phosphorylation, in LPS-induced inflammation. METTL3 knockdown promoted the mRNA expression and stability of negative regulators of Smad signaling, Smad7 and Smurf1, the same regulatory pattern identified when the m6A-binding protein YTHDF2 was silenced. Moreover, METTL3 depletion enhanced proinflammatory cytokine expression and increased the phosphorylation of ERK, p38, JNK, and p65 in MAPK and NF-κB signaling pathways. The increase in cytokine expression was inhibited after MAPK signaling inhibitor treatment. All data suggest that METTL3 knockdown inhibits osteoblast differentiation and Smad-dependent signaling by stabilizing Smad7 and Smurf1 mRNA transcripts via YTHDF2 involvement and activates the inflammatory response by regulating MAPK signaling in LPS-induced inflammation.

Highlights

Bone is a highly adaptive tissue with remodeling processes coordinated by bone-forming osteoblasts and bone-resorbing osteoclasts
The results showed that the mRNA expression of the osteogenic markers Runx2, Sp7, Alpl, and Col1a1 and the protein levels of RUNX2 and OSTERIX significantly increased (Figure 1C,D)
We found that ERK, p38, JNK, and p65 phosphorylation in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways was enhanced in methyltransferase-like 3 (METTL3)-knockdown cells, further confirming the role of METTL3 as a negative regulator of the LPS-induced inflammatory response in osteoblasts

Summary

Introduction

Bone is a highly adaptive tissue with remodeling processes coordinated by bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts have osteogenic differentiation potential and are responsible for mediating bone metabolism through synthesizing and depositing extracellular matrix [1,2,3]. BMP/Smad signaling is fundamentally important in bone homeostasis, and canonical Smad-dependent signaling exhibits pivotal regulatory functions in osteogenic gene expression by phosphorylating the Smad1/5/9 complex [4]. Lipopolysaccharide (LPS) from the cell membrane of gram-negative bacteria is recognized as the main pathogenic factor positively related to infectious bone destruction. It has been reported that LPS can suppress osteoblastic differentiation by affecting multiple pathways, such as BMP/Smad signaling, Wnt/β-catenin signaling, and Notch signaling [8,9,10]. LPS stimulates osteoblasts to produce various cytokines by activating mitogen-activated protein kinase (MAPK) signaling and nuclear factor-κB (NF-κB) signaling, contributing to the global systemic and local inflammatory responses [11,12]

Methods

Results

Discussion

Conclusion