Methylglyoxal – an advanced glycation end products (AGEs) precursor – inhibits differentiation of human MSC-derived osteoblasts in vitro independently of receptor for AGEs (RAGE)

Waqas, Komal; Muller, Max; Koedam, Marijke; el Kadi, Youssra; Zillikens, M. Carola; van der Eerden, B. C. J.

Affiliations

¹Department of Internal Medicine, Erasmus MC, Erasmus University Medical Center, Rotterdam, the Netherlands

Abstract and keywords

A major precursor of advanced glycation end-products (AGEs) - methylglyoxal (MG) - is a reactive carbonyl metabolite that originates from glycolytic pathways. MG formation and accumulation has been implicated in the pathogenesis of diabetes and age-related chronic musculoskeletal disorders. Human bone marrow-derived stromal cells (BMSCs) are multipotent cells that have the potential to differentiate into cells of mesenchymal origin including osteoblasts, but the role of MG on their differentiation is unclear. We therefore evaluated the effect of MG on proliferation and differentiation of BMSC-derived osteoblasts.

Cells were treated with different concentrations of MG (600, 800 and 1000 μM). Cell viability was assessed using a Cell Counting Kit-8 assay. Alkaline phosphatase (ALP) activity and calcium deposition assays were performed to evaluate osteoblast differentiation and mineralization. Gene expression was measured using qRT-PCR, whereas AGE specific receptor (RAGE) and collagen 1 were examined by immunocytochemistry and Western blotting. RAGE knockdown was performed by transducing RAGE specific short hairpin RNAs (shRNAs) using lentivirus.

During osteogenic differentiation, MG treatment resulted in reduction of cell viability (27.7 %), ALP activity (45.5 %) and mineralization (82.3 %) compared to untreated cells. MG significantly decreased expression of genes involved in osteogenic differentiation - RUNX2 (2.8 fold), ALPL (3.2 fold), MG detoxification through glyoxalase - GLO1 (3 fold) and collagen metabolism - COL1A1 (4.9 fold), COL1A2 (6.8 fold), LOX (5.4 fold) and PLOD1 (1.7 fold). MG significantly reduced expression of collagen 1 (53.3 %) and RAGE (43.1 %) at protein levels. Co-treatment with a MG scavenger - aminoguanidine – prevented all negative effects of MG. RAGE-specific knockdown during MG treatment did not reverse the effects on cell viability, osteogenic differentiation or collagen metabolism.

In conclusion, MG treatment can negatively influence the collagen metabolism and differentiation of BMSCs-derived osteoblasts through a RAGE independent mechanism.

Keywords: Advanced glycation end products; Methylglyoxal; Osteoblasts; Mesenchymal stem cells; Receptor for AGEs

Links

DOI: 10.1016/j.bone.2022.116526

PubMed: 35995334

WoS: 000863197700002

History

Received: 2022-05-17

Revised: 2022-07-27

Accepted: 2022-08-16

Online: 2022-08-19

Cited Works (4)

Year	Entry
2020	Arakawa S, Suzuki R, Kurosaka D, Ikeda R, Hayashi H, Kayama T, Ohno R-i, Nagai R, Marumo K, Saito M. Mass spectrometric quantitation of AGEs and enzymatic crosslinks in human cancellous bone. Sci Rep. 2020;10:18774.
2010	Tang SY, Vashishth D. Non-enzymatic glycation alters microdamage formation in human cancellous bone. Bone. January 2010;46(1):148-154.
2020	Suzuki R, Fujiwara Y, Saito M, Arakawa S, Shirakawa J, Yamanaka M, Komohara Y, Marumo K, Nagai R. Intracellular accumulation of advanced glycation end products induces osteoblast apoptosis via endoplasmic reticulum stress. J Bone Miner Res. October 2020;35(10):1992-2003.
2020	Waqas K, Chen J, Koromani F, Trajanoska K, van der Eerden BCJ, Uitterlinden AG, Rivadeneira F, Zillikens MC. Skin autofluorescence, a noninvasive biomarker for advanced glycation end‐products, is associated with prevalent vertebral and major osteoporotic fractures: the Rotterdam Study. J Bone Miner Res. October 2020;35(10):1904-1913.