Fig 1: Cellular adaptive signaling pathways activated by cold exposure. RBM3 activation and downstream osteogenic potential following short-term cold exposure. (A) Cold exposure triggers a cellular cold-shock response, activating RBM3 via HSPs released by phosphorylated and nuclear-localized HSF1. (B) HSP70 activates TLR4, initiating the MyD88/NF-kB pathway, with phosphorylated NF-kB essential for RBM3 expression. (C) RBM3 activates the MAPK/ERK pathway, leading to Runx2 and osteocalcin (OCN) expression, crucial for osteoblast differentiation. PGC-1a activation and downstream osteogenic potential following short-term cold exposure. PGC-1α activation and osteogenic potential following short-term cold exposure. (D) Cold activation of adrenergic receptors and TRPs stimulates cAMP and calcium influx, leading to CREB phosphorylation via the PKA/Ca2+/CaMK pathway. (E) PGC-1α forms a complex with ERRα in osteoblastic cells, promoting differentiation via the Runx2/OCN pathway for mineralization. (F) PGC-1α and NRF-1/NRF-2 activate TFAM and NCMP, crucial for mitochondrial biogenesis and intracellular calcium storage.
Fig 2: Vhl deletion in osteoblasts promotes the expression of RegIIIγ.a Ocn mRNA expression in the bone tissue of the Vhl cKO mice and their littermate controls (n = 4). b Levels of OCN in the serum of the Vhl cKO mice and their littermate controls (n = 5). c KEGG enrichment analysis of differentially expressed mRNAs between the bone tissue of the Vhl cKO mice and their littermate controls (n = 4). d Heatmap of mRNA expression in the bone tissue of the Vhl cKO mice and their littermate controls (n = 4). e RT‒qPCR analysis of RegIIIγ mRNA expression in vitro in the Vhlflox/flox osteoblasts infected with Cre or control adenovirus (n = 3). f WB analysis of RegIIIγ expression in vitro in the Vhlflox/flox osteoblasts infected with Cre and control adenoviruses. g IF staining showing RegIIIγ expression in vitro in the Vhlflox/flox osteoblasts infected with Cre and control adenoviruses. h ELISA analysis of the in vitro level of secreted RegIIIγ in the Vhlflox/flox osteoblasts infected with Cre and control adenoviruses (n = 5). i, j Representative images of RegIIIγ IHC and IF staining of bone tissue from the Vhl cKO mice and their littermate controls. k Increased RegIIIγ mRNA expression was also found in the bone tissues of the Vhl cKO mice. l Cytokine microarray analysis of differentially expressed proteins in the serum of the Vhl cKO mice and their littermate controls (n = 4). m Differential enrichment analysis of serum cytokines between the Vhl cKO mice and their littermate controls (n = 4). n Serum RegIIIγ levels in the Vhl cKO mice and their littermate controls (n = 5). o Expression of RegIIIγ after Hif-1α or Hif-2α knockout in the Vhl knockout osteoblasts. p Dual-luciferase reporter assays to identify the Hif-1α-induced increase in RegIIIγ promoter activity. q ChIP‒qPCR analysis of the binding site of HIF-1α to the RegIIIγ promoter. All data are presented as the mean ± SEM, and p values were analyzed by two-tailed t-tests for (a, b, e, h, k, n, q) and one-way ANOVA for (p). ns, with no significant difference, **p < 0.01, ***p < 0.001. All the data are representative of two to three independent experiments. The data were obtained from male mice.
Fig 3: Detection of ALP and OCN at day 5 and day 10 within isolated osteoblasts following cold exposure and mineralization within isolate osteoblasts following cold exposure for 14 days. (A) ALP activity after 5 days of cold exposure was 2.72 U/L (µmol/min/L) ± 0.85, while in the non-treated group it was 0.74 U/L ± 0.35 (* p-value = 0.021). (B) ALP activity after 10 days of cold exposure was 2.25 U/L ± 0.04, while in the non-treated group it was 1.0301 U/L ± 0.24 (** p-value < 0.001). (C) OCN levels after 5 days of cold exposure was 0.34 mg/mL ± 0.10, while in the non-treated group it was 0.43 mg/mL ± 0.10 (p-value = 0.29). (D) OCN levels after 10 days of cold exposure was 0.84 mg/mL ± 0.17, while in the non-treated group it was 0.35 mg/mL ± 0.10 (*** p-value = 0.019). ARS (red) detection within isolated osteoblasts (10× magnification). Scale bar represents 250 µm. (E) Osteoblasts isolated from non-treated fractured femurs. (F) Osteoblasts isolated from daily cold-treated fractured femurs. (G) ARS staining analysis: Detection of ARS after 14 days of cold exposure was 1.04% ± 0.40, while in the non-treated group it was 0.27% ± 0.025 (*** p-value = 0.030).
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