Fig. 9: The nuclear transcription factor PPARα mediates T2D-induced specific reduction in the expression of osteocytic SERCA2 pump.

a RNA-seq-based KEGG pathway analysis showing the top 10 enriched KEGG pathways. b, c GSEA analysis showing a significant enrichment of signaling events associated with PPARα and PPARDR1_Q2. d The PPARα and p-PPARα protein expression in osteocytes. e Immunohistochemical staining of osteocytic PPARα in diabetic and non-diabetic tibiae. f The SERCA2 expression in osteocytes treated with antagonists of PPARα (MK886 and GW6471), PPARβ/δ (GSK0660 and GSK3787), and PPARγ (T0070907 and GW9662). g The SERCA2 expression in HGHF-exposed osteocytes treated with agonists of PPARα (Fenofibrate and GW7647), PPARβ/δ (GW0742 and GW501516), and PPARγ (rosiglitazone and pioglitazone). h A schematic representation and the relative luciferase activities of three ATP2A2 promotor regions. i The relative luciferase activity assays of Seg#1, Seg#2 and Seg#3. j The relative luciferase activity in HGHF-treated osteocytes with PPARα silencing. k ChIP assays showing the PPARα enrichment on ATP2A2 promotor in normal and HGHF-treated osteocytes. l EMSA assays confirming the binding of PPARα to the ATP2A2 promoter region (−620 to −608 bp). The nuclear extract were incubated with biotin-labeled wild-type (WT-biotin) probe, unlabeled wild-type (WT) probe, and biotin-labeled mutated (Mut-biotin) probe. Red letters indicate substituted nucleotide sequences in the mutated probes (P1: −620 to −608 bp; P2: −1283 to −1277 bp). m, n Intracellular Ca²⁺ signaling and protein expression of osteocyte-related cytokines in HGHF-treated osteocytes with PPARα overexpression subjected to FSS. o, p Intracellular Ca²⁺ signaling and the expression of osteocyte-related cytokines in MLO-Y4 cells with lentiviral silencing of PPARα subjected to FSS. Graphs represent mean ± SD (d, f, g, i–k, m, o n = 6 biologically independent replicates; e n = 8 mice per group; l n = 3 independent replicates; n, p: n = 120 cells per group). a P value was obtained by one-tailed hypergeometric test. b, c P values were obtained by one-tailed permutation test. d–h, n, p ***P < 0.001 by one-way ANOVA with Bonferroni’s post test. i–k ***P < 0.001 by two-way ANOVA with Bonferroni’s post test. Specific P values are provided in the Source Data file. Scale bars: e 20 μm.