Metabolic changes in synovial cells in early inflammation: Involvement of CREB phosphorylation in the anti-inflammatory effect of 2-deoxyglucose

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Abstract

The involvement of metabolic reprogramming has been suggested to contribute to the pathophysiology of rheumatoid arthritis (RA). Glycolysis is enhanced in synovial cell metabolism in RA patients. Inhibitors of glycolysis are known to have anti-inflammatory effects. But, changes in the metabolism of normal synovial membranes or synovial cells during the early stages of inflammation remains unknown. Moreover, there are still many aspects of inflammatory signaling pathways altered by glycolysis inhibitors, that remain unclear. In this study we found that, in normal, non-pathological bovine synovial cells, most of ATP synthesis was generated by mitochondrial respiration. However, during the early of stages inflammation, initiated by lipopolysaccharide (LPS) exposure, synovial cells shifted to glycolysis for ATP production. The glycolysis inhibitor 2-deoxyglucose (2DG) reversed LPS induced increases in glycolysis for ATP production and suppressed the expression of inflammatory cytokines and proteolytic enzymes. 2DG suppressed the phosphorylation of the transcription factor cAMP response element binding protein (CREB) enhanced by LPS. Treatment with a CREB inhibitor reversed the expression of LPS-stimulated inflammatory cytokines and proteolytic enzymes. This study showed that changes in metabolism occur during the early stages of inflammation of synovial cells and can be reversed by 2DG and signaling pathways associated with CREB phosphorylation.

Introduction

Rheumatoid arthritis (RA) is characterized by the inflammation and proliferation of synovium, leading to the destruction of articular cartilage and bone [1]. Recently treatment of RA has improved dramatically, and many patients reach a state of remission with few symptoms. On the other hand, the etiology of RA has not yet been clarified [2], and no treatment methods has been established that can completely cure the disease. In addition, it has been reported that about 5–20% of RA patients remain are difficult to control with the current treatment [3]. Furthermore, it is difficult to discontinue the anti-rheumatic drug even after RA is in remission [4]. Other major problems include the increased risk of complications due to use of anti-rheumatic drugs [5,6] and the long-term financial burden of expensive drugs [7]. Thus, it is desired to develop treatment methods that target a mechanism different from that of current treatment.

In recent years, the involvement of metabolic reprogramming has been suggested to play an important role in the pathophysiology of RA and thus, metabolic pathways have been reported to be a new therapeutic target for RA [[8], [9], [10]]. Synovial proliferation is observed in RA joints, and inflamed synovial cells undergo metabolic changes similar to those observed in tumor cells (Warburg effect), both resulting in cell proliferation [9,11]. Cells in the body utilize nutrients such as glucose and amino acids to produce the energy storing molecule adenosine triphosphate (ATP). When breaking down glucose to produce energy, aerobic glycolysis pathways are typically used -pathways that consume oxygen and produce ATP by oxidative phosphorylation of mitochondria [12]. On the other hand, in tumor cells and RA synovial cells, anaerobic glycolysis pathway come into play, producing ATP without consuming oxygen [13]. It has been reported that glycolysis is enhanced in synovial cells collected from RA patients, and that glycolysis inhibitors have anti-inflammatory effects [8,14].

Previous studies concerning synovial cell metabolism have primarily examined human synovial cells collected during artificial joint replacement [8]; that is, synovial cells associated with late stage RA for experiments. There are no reports on changes in the metabolism of normal synovial membranes or synovial cells in the early stages of inflammation. In addition, there are still many unclear points about the signaling pathways in synovial cells [9] that are affected by the anti-inflammatory effects of glycolysis inhibitors.

The purpose of this study is to investigate the intracellular metabolism of the synovial cells in the normal or early stage of inflammation. In addition, we also investigated the signaling pathways of anti-inflammatory effect of glycolysis inhibitors on synovial cells.

Section snippets

Methods

Materials. Dulbecco's modified Eagle's medium (DMEM) were obtained from Thermo Fischer Scientific. Trypsin ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (Pasching, Austria). Collagenase P (Cat. No.:11249002001; Roche Applied Science) were used in the dissociation of tissues. Lipopolysaccharide (Cat. No.:437628) was obtained from Sigma-Aldrich. 2-deoxyglucose (Cat. No.:24894205) was obtained from

Results

Changes of intracellular metabolism in synovial cell during early inflammation. Changes to metabolism during early inflammation were first explored by monitoring changes in gene expression of two key proteins, glucose transporter-1 (GLUT1) and hexokinase-2 (HK2). Primary bovine synovial cell cultures were treated with varying concentrations of LPS for 12 h and the expression of GLUT1 and HK2 mRNA quantified by qRT-PCR. GLUT1 mRNA expression was increased at LPS concentrations as low as10 ng/ml,

Discussion

In recent years, the involvement of metabolic reprogramming has been suggested as key to the pathophysiology of RA and thus, pathways related to cellular metabolism become new, potential therapeutic targets for RA [[8], [9], [10]]. In addition, metabolomic studies may provide relevant biomarkers to improve diagnostic accuracy, definition of prognosis and monitoring the efficacy of the treatment in rheumatoid arthritis [20]. Recently several studies reported that glycolysis inhibitors have

Conclusions

We confirmed that changes in intermediary metabolism are closely associated with the early stages of inflammation in non-pathological, bovine synovial cells. While most ATP synthesis is supplied by mitochondrial respiration in normal synovial cells, the metabolism shifts to a heavier reliance on the glycolysis pathway for ATP production as one of the earliest inflammation events. Additionally, we showed that the signaling pathways of anti-inflammatory effect of glycolysis inhibitors in synovial

Author contributions

K.T. and N.T. contributed to the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting/revising the manuscript critically for important intellectual content, and provided final approval of the version to be submitted. T.K. and S.I. contributed to the conception and design of the study, acquisition of data, revising the manuscript critically for important intellectual content, and provided final approval of the version to be submitted. Y. Y.,

Data availability

All data generated or analyzed during this study are includes in this published article.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgments

The work was supported in part by JSPS KAKENHI Grant Number JP18K16678, 19K09620, 19K09619 (KT, NT and TK). The authors also thank Dr. Kinji Ohno (Director) and Dr. Mikako Ito (Division of Neurogenetics, Nagoya University Graduate School of Medicine) for usage of Seahorse flux analyzer. The authors also thank Dr. Warren Knudson for valuable comments to this work and proofreading English. The authors wish to acknowledge Division for Medical Research Engineering, Nagoya University Graduate School

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