Methylation of 4-aminobutyrate aminotransferase (ABAT) by dnmy3b regulates chondrocytes metabolism and the development of osteoarthritis (OA)

Abstract

Purpose: Our recent work established that loss of Dnmt3b in chondrocytes leads to OA progression, at least in part, due to accelerated TCA cycle and elevated mitochondrial respiration. The goal of this study is to determine the downstream targets of Dnmt3b potentially involved in regulating chondrocyte energy metabolism and in the pathogenesis of OA. Methods: Primary articular chondrocyte (AC) isolation and culture: ACs were isolated from 2-month-old Dnmt3b f/f mice following with Ad-Cre virus to delete Dnmt3b gene. Abat siRNA, Lenti Abat and Vigabatrin were used to regulate Abat expression and activity. Whole genome bisulfite sequencing (Methyl-seq) and RNA-seq: Genomic DNA and RNA were isolated from Dnmt3b loss-of-function (LOF) ACs for detailed gene expression and methylation profile analyses. HPLC: TCA metabolites were measured by HPLC, including succinate and fumarate. Mito stress assay: Mitochondrial function was assessed by seahorse mito stress test to measure basal and maximal oxygen consumption rate (OCR) as well as ATP production. Meniscal Ligament Injury (MLI): MLI surgeries were performed on 10-week-old wild type mice to induce OA progression. Vigabatrin was given by i.p injection every other day for 6 or 10 weeks. Results: Increased Abat expression in OA chondrocytes. RNA-seq and Methyl-seq were performed to investigate the downstream targets of Dnmt3b and integrative analysis revealed an enrichment in genes and pathways that regulate cellular energy metabolism in Dnmt3b LOF cells. Among these gene targets was 4-aminobutyrate aminotransferase (Abat). Direct analysis of the Abat promoter region confirmed that Dnmt3b LOF resulted in reduced methylation in ACs (50% methylation in Control; and 3% methylation in Dnmt3b LOF. qPCR and Western blot further showed that both Abat gene and protein expression were induced in Dnmt3b LOF ACs and OA patients. Abat enhances mitochondrial oxidative phosphorylation (OXPHOS) and chondrocyte hypertrophy. Since Abat catalyzes the metabolism of GABA to succinate, HPLC mass spectroscopy validated an increase in both succinate (300%) and fumarate (150%) levels in Dnmt3b LOF cells. We further evaluated if Abat is involved in regulation of OXPHOS and AC hypertrophy and found that Abat siRNA resulted in a decrease in OXPHOS as reduced basal OCR (50%), maximal OCR (60%), and ATP production (50%). Abat inhibition in Dnmt3b LOF ACs by Vigabatrin blocked the increase in the expression of Col10a1, Mmp13, and Runx2 observed in the Dnmt3b LOF chondrocytes. In contrast, Abat overexpression led to a marked increase in basal OCR (300%), maximal OCR (500%), and ATP production (500%). Over-expression of Abat stimulated chondrocyte hypertrophy as reflected by upregulation of Runx2 (250%) and Mmp13 (600%). To link the Abat mediated increase of TCA metabolites as a mechanism regulating OXPHOS, we added 1mM succinate to WT AC cultures and found an increase in hypertrophic chondrocyte genes. In addition, succinate treatment increased the basal OCR (150%), maximal OCR (150%), and ATP production (200%). Abat inhibition attenuates OA progression in mice following knee injury: MLI surgeries were performed on 10-week-old male WT mice and vehicle treated mice developed advanced osteoarthritic changes with loss of cartilage, development of osteophytes, and subchondral bone thickening. However, mice treated with Vigabatrin displayed a protective effect against injury-induced OA progression at 6 and 10 weeks post MLI surgery. OARSI scoring showed marked lower scores in Vigabatrin treated mice. Conclusions: Our previous work has established that loss of Dnmt3b stimulated chondrocyte hypertrophy and resulted in increased OXPHOS, with increases in TCA metabolites, NADH, and basal respiration and maximal O2 capacity. Through integration of RNA-seq and Methyl-seq data, we now show that Abat is a target of Dnmt3b, and further establish a critical role for energy metabolism in the development of OA.