Journal Article

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells

Kim, Kyoung-Sook, Cho, Jong-Hyun, Kim, Cheorl-Ho, Kim, Hee-Do, Lee, Young-Choon, An, So-Young, Kim, Hyeon-Jun, Yoon, Hyun-Kyoung

2023

Overview

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase ( hST8Sia I ) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Activator protein 1 / Analysis / Antibodies / Binding sites / Biology and Life Sciences / Biosynthesis / Bone cancer / Cell activation / Cell differentiation / Chromatin / Cyclic AMP response element-binding protein / Diagnosis / Differentiation (biology) / DNA binding proteins / Gangliosides / GD3 synthase / Gene expression / Genes / Genetic aspects / Genetic transcription / Immunofluorescence / Immunoprecipitation / Luciferase / NF-κB protein / Osteoblastogenesis / Osteocalcin / Osteosarcoma / Plasmids / Prostate cancer / Proteins / Research and Analysis Methods / Sarcoma / Signal transduction / Site-directed mutagenesis / Starvation / Transcription activation / Transcription factors / Up-regulation