JKAMP inhibits the osteogenic capacity of adipose-derived stem cells in diabetic osteoporosis by modulating the Wnt signaling pathway through intragenic DNA methylation

Abstract

Background: Diabetic osteoporosis (DOP) is a systemic metabolic bone disease caused by diabetes mellitus (DM). Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Our previous study confirmed that ASCs from DOP mice (DOP-ASCs) have a lower osteogenesis potential compared with control ASCs (CON-ASCs). However, the cause of this poor osteogenesis has not been elucidated. Therefore, this study investigated the underlying mechanism of the decline in the osteogenic potential of DOP-ASCs from the perspective of epigenetics and explored methods to enhance their osteogenic capacity.

Methods: The expression level of JNK1-associated membrane protein (JKAMP) and degree of DNA methylation in CON-ASCs and DOP-ASCs were measured by mRNA expression profiling and MeDIP sequencing, respectively. JKAMP small interfering RNA (siRNA) and a Jkamp overexpression plasmid were used to assess the role of JKAMP in osteogenic differentiation of CON-ASCs and DOP-ASCs. Immunofluorescence, qPCR, and western blotting were used to measure changes in expression of Wnt signaling pathway-related genes and osteogenesis-related molecules after osteogenesis induction. Alizarin red and ALP staining was used to confirm the osteogenic potential of stem cells. Bisulfite-specific PCR (BSP) was used to detect JKAMP methylation degree.

Results: Expression of JKAMP and osteogenesis-related molecules (RUNX2 and OPN) in DOP-ASCs was decreased significantly in comparison with CON-ASCs. JKAMP silencing inhibited the Wnt signaling pathway and reduced the osteogenic ability of CON-ASCs. Overexpression of JKAMP in DOP-ASCs rescued the impaired osteogenic capacity caused by DOP. Moreover, JKAMP in DOP-ASCs contained intragenic DNA hypermethylated regions related to the downregulation of JKAMP expression.

Conclusions: Intragenic DNA methylation inhibits the osteogenic ability of DOP-ASCs by suppressing expression of JKAMP and the Wnt signaling pathway. This study shows an epigenetic explanation for the reduced osteogenic ability of DOP-ASCs and provides a potential therapeutic target to prevent and treat osteoporosis.

Keywords: Adipose-derived stem cells; DNA methylation; Diabetic osteoporosis; JKAMP; Osteogenic differentiation; Wnt signaling pathway.

Fig. 1

JKAMP, the Wnt signaling pathway, and osteogenesis-related molecules are suppressed in DOP-ASCs. a Normal appearance passage 2 CON-ASCs and DOP-ASCs. b mRNA levels of Jkamp, Jnk1, β-catenin, Runx2, and Opn in CON-ASCs and DOP-ASCs. c Protein levels of JAKMP, Wnt signaling pathway-related molecules, and osteogenesis-related molecules in CON-ASCs and DOP-ASCs. Data represent the mean ± SD of at least three independent experiments, *P < 0.05, **P < 0.01

Fig. 2

Si-Jkamp suppresses Wnt signaling and osteogenesis-related molecules in CON-ASCs (osteoinduction for 3 days). a, b Si-Jkamp downregulated the gene and protein levels of Wnt signaling pathway markers and osteogenesis-related molecules in CON-ASCs (osteoinduction for 3 days). Data represent the mean ± SD of at least three independent experiments, *P < 0.05, **P < 0.01

Fig. 3

Si-Jkamp suppresses Wnt signaling and osteogenesis-related molecules in CON-ASCs (osteoinduction for 6 days). a, b mRNA and protein levels of Wnt signaling pathway markers and osteogenesis-related molecules were downregulated after si-Jkamp transfection into CON-ASCs (osteoinduction for 6 days). Data represent the mean ± SD of three or more independent experiments, *P < 0.05, **P < 0.01

Fig. 4

Si-Jkamp decreases the osteogenic ability of CON-ASCs. a, b Immunofluorescence staining of RUX2 and OPN proteins in CON-ASCs after 3 days after osteoinduction. c Alizarin red staining of CON-ASCs after 14 days of osteoinduction. d, e ALP staining of CON-ASCs after 3 and 5 days of osteoinduction. d Osteoinduction for 3 days. e Osteoinduction for 5 days

Fig. 5

Overexpression of Jkamp increases Wnt signaling and osteogenesis-related molecules in DOP-ASCs (osteoinduction for 3 days). a, b The Jkamp plasmid upregulated mRNA and protein levels of Wnt signaling pathway markers and osteogenesis-related molecules in DOP-ASCs (osteoinduction for 3 days). c DOP-ASCs cellular uptake of NC- plasmids and OE- plasmids after treated for 48 h. Data represent the mean ± SD of at least three independent experiments, *P < 0.05, **P < 0.01

Fig. 6

Overexpression of Jkamp increases Wnt signaling and osteogenesis-related molecules in DOP-ASCs (osteoinduction for 6 days). a, b mRNA and protein levels of Wnt signaling pathway markers and osteogenesis-related molecules were upregulated after Jkamp plasmid transfection into DOP-ASCs (osteoinduction for 6 days). Data represent the mean ± SD of at least three independent experiments, *P < 0.05, **P < 0.01

Fig. 7

Overexpression of Jkamp increases the osteogenic ability of DOP-ASCs. a, b Immunofluorescence staining of RUX2 and OPN proteins in DOP-ASC after 3 days of osteoinduction. c Alizarin red staining of DOP-ASCs after 14 days of osteoinduction. d, e ALP staining of DOP-ASCs after 3 and 5 days of osteoinduction. c Osteoinduction for 3 days. d Osteoinduction for 5 days

Fig. 8

Increased methylation level of JKAMP in DOP-ASCs. a MeDIP sequencing showed that the intragenic methylation peaks of the JKAMP gene in DOP-ASCs were significantly higher than those in CON-ASCs. b Meth Primer software analysis showed that a large amount of CGI was enriched in the region near JKAMP exon 4 (genomic coordinates: chr12, 72093357–72094707). c, d BSP confirmed that the methylation degree of CON-ASCs was lower than that of matched DOP-ASCs in the region near exon 4 of JKAMP (genomic coordinates: chr12, 72093857–72094207). e Correlation between Jkamp mRNA levels and MeDIP peak enrichment near exon 4 of JKAMP (r² = 0.977). Each data point corresponds to a high-throughput sequencing result, and the data were analyzed for correlation. Data represent the mean ± SD of at least three independent experiments, *P < 0.05, **P < 0.01