Expression of RUNX2/LAPTM5 in the Induction of MC3T3-e1 Mineralization and Its Possible Relationship with Autophagy

Abstract

Background: The study aims to correlate osteogenesis with autophagy during the mineralization induction of MC3T3-e1 through exploring the expression of runt-related transcription factor 2 (RUNX2)/lysosomal-associated transmembrane protein 5 (LAMPT5).

Methods: The induction of mineralization in MC3T3-e1 was followed by detecting the expressions of osteogenesis-related indexes such as RUNX2, alkaline phosphatase (ALP), osteocalcin (OCN), and LAPTM5 using RT-qPCR and Western blot from 0 to 14 days. Transmission electron microscope was utilised in visualizing the alterations of autophagosomes, which was followed by immunofluorescence detecting the subcellular localization of autophagy-related index sequestosome 1 (P62) and microtubule-associated protein 1 light 3 (LC3) protein and scrutinising the expression of P62 mRNA and P62 and LC3 proteins.

Results: Induction of MC3T3-e1 mineralization demonstrated an increased expression of osteogenesis-related indicators such as RUNX2, ALP, OCN, and LAPTM5 (p < 0.05), as evident from the results of RT-qPCR and Western blot. Meanwhile, the expression of autophagosomes increased one day after mineralization induction and then experienced a gradual decline, and enhanced expression of LC3 protein was noted on days 1-2 of mineralization induction but was then followed by a corresponding reduce. In contrast, a continuous increase was reported in the expression of P62 mRNA and protein, respectively (p < 0.05). Up- and down-regulating RUNX2/LAPTM5 expression alone confirmed the aforementioned results.

Conclusion: It was therefore proposed that RUNX2 may be responsible for an early increase and then a gradual decrease in LAPTM5-mediated autophagy through the regulation of its high expression. Meanwhile, increased LAPTM5 expression in osteogenic mineralization presumed that RUNX2/LAPTM5 promoted autophagy and osteogenic expression, which may play a bridging role in the regulation of autophagy and osteogenesis.

Keywords: Autophagy; Correlativity; LAPTM5; Osteogenesis; RUNX2.