Primary Osteocyte Supernatants Metabolomic Profiling of Two Transgenic Mice With Connexin43 Dominant Negative Mutants.

Baoqiang Yang,Meng Chen,Kaiting Ning,Huiyun Xu,Jean X Jiang,Dong-En Wang,Guobin Li,Lan Zhang

doi:10.3389/fendo.2021.649994

Abstract

Osteocytes could release some small molecules (≤ 1 kDa) through gap junctions and hemichannels to extracellular environment, such as prostaglandin E2 (PGE2), nitric oxide (NO) and adenosine triphosphate (ATP), which play key roles in transferring signals between bone cells and other tissue cells. Connexin (Cx) 43 is the most abundant connexin in osteocytes. To further discover molecules released by osteocytes through Cx43 channels and better understand the regulatory function of Cx43 channels in osteocytes, we performed non-targeted global metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on conditioned medium collected from osteocytes isolated from two transgenic mouse models with Cx43 dominant negative mutants driven by a 10 kb-DMP1 promoter: R76W (gap junctions are blocked, whereas hemichannels are promoted) and Δ130-136 (both gap junctions and hemichannels are blocked). The results revealed that several new categories of molecules, such as “fatty acyls” and “carboxylic acids and derivatives”, could be released through osteocytic Cx43 channels. In addition, alteration of Cx43 channel function affected the release of metabolites related to inflammatory reaction and oxidative stress. Pathway analysis further showed that citric acid cycle was the most differential metabolic pathway regulated by Cx43 channels. In sum, these results isolated new potential metabolites released by osteocytes through Cx43 channels, and offered a novel perspective to understand the regulatory mechanisms of osteocytes on themselves and other cells as well.

Highlights

The osteocyte is the most abundant cell type (90% - 95%) in bone tissue [1]
To evaluate the capability of the LC-MS/ MS-based metabolomics approach performed in the study, we first analyzed the total ion chromatograms of quality control (QC) samples
connexin 43 (Cx43) channels participate in regulating global metabolism through mediating the release of small molecules, such as adenosine triphosphate (ATP), IP3, prostaglandin E2 (PGE2), glutathione, amino acids and cyclic nucleotides [27,28,29]

Summary

Introduction

The osteocyte is the most abundant cell type (90% - 95%) in bone tissue [1]. Connexin 43 (Cx43), the most abundant connexin expressed in osteocytes [7], forms gap junctions and hemichannels that serve as physical linkages to allow the exchange of small molecules (≤ 1 kDa) between adjacent cells and extracellular environment [8]. Gap junctions and hemichannels play pivotal roles in regulating signal transduction between osteocytes and other bone cells to participate in the modeling and remodeling of the bone [9], and between osteocytes and other tissue cells to regulate the function of muscle, kidney, and fat metabolism [10,11,12]. More molecules are still not clear and needed to be discovered, which are essential to further understand the molecular mechanisms underlying osteocytemediated effects on bone homeostasis and other systemic diseases

Methods

Results

Conclusion