The volcano plot compares gene expression between Day 0 – 24 h – 0 nM and Day 0 – 24 h – 100 nM conditions in undifferentiated MLO-A5 cells treated with a high dose of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We found the upregulation of Spp1 (osteopontin) and Mmp13 (matrix metallopeptidase), suggesting a role in extracellular matrix remodeling and osteoblast activity. Conversely, downregulated genes like Dgat1 and Heg1 may reflect suppression of metabolic pathways. Additionally, genes involved in mineralization and growth factor regulation, such as Mgp and Sulf2, align with the study’s focus on osteocyte-like cells. The results are filtered for statistical significance (padj < 0.05, |log2FC| > 1), and duplicate labels, like Lgals2, may require further verification. In the following experiments, we can focus on genes with large levels of upregulation or downregulation.

The PCA plot shows a clear separation between d0_24h_100nm and d0_24h_0nm conditions, with the primary variation driven by treatment (85.5% variance along PC1). This strong segregation indicates distinct transcriptional responses to the high dose of 1,25(OH)₂D₃. The minimal variation along PC2 (5.5%) suggests that other factors, such as biological replicates, contribute less to the overall differences. 

We compared the expression levels of four genes—Vdr, Cyp24a1, Spp1, and Enpp1—under two conditions: d0_24h_0nm and d0_24h_100nm. The results reveal consistent upregulation of all four genes in response to the 100 nM treatment. 

Vdr (Vitamin D receptor) expression showed a substantial increase following treatment, with normalized expression levels rising from a relatively low baseline in the control group. This suggests that the treatment activates pathways involving Vdr.

Cyp24a1, an enzyme that plays a role in vitamin D metabolism, exhibited the most dramatic change. Expression was nearly undetectable in the control group but significantly elevated under the 100 nM condition. This strong induction highlights a potential regulatory effect of the treatment on vitamin D catabolism.

Spp1, also known as osteopontin, was similarly induced. Its expression increased by several orders of magnitude, indicating a robust response to the treatment and possible involvement in mineralization or immune-related processes.

Enpp1 expression was also upregulated, though to a lesser extent than Spp1 or Cyp24a1. Nonetheless, the consistent increase in expression under treatment conditions suggests that Enpp1 may be part of the downstream response. 

Overall, these findings demonstrate that the 100 nM treatment condition strongly influences gene expression in this system, particularly for genes related to vitamin D signaling and mineral metabolism.