Lysyl oxidase (LOX) dependent cross-linking is deficient in diabetic bone. Here we investigate regulation of LOX by Wnt3a and TNF-α in a mesenchymal stem cell line as a model for a mechanism of diabetic osteopenia.
Method:
C3H10T1/2 cells were treated with Wnt3a +/- TNF-α. LOX expression (qPCR), promoter/reporter activity, and mRNA stability were measured. In silico analyses identified three potential cis-elements in the LOX promoter mediating effects of Wnt3a, which were then evaluated experimentally after site directed mutagenesis. A PCR microarray (miR) screen (Qiagen/SA Biosciences) then identified miRs which regulate LOX mRNA stability in response to TNF-α. The functionality of identified miRs was validated using miRNA mimics, and miRNA reporter assays.
Result:
Wnt3a up-regulated LOX 3.5-fold at 135 ng/ml. In silico analyses of the murine LOX promoter identified three Wnt3a-responsive TCF/LEF-binding cis-elements which were next evaluated experimentally by site-directed mutagenesis. The single element located at -1225 to -1214 nucleotides proximal to the translation start site mediated Wnt3a-induced LOX transcription. TNF-α (20 ng/ml) blocked Wnt3a up-regulation of LOX. TNF-α did not inhibit LOX transcription, but decreased the stability of LOX mRNA determined in Wnt3a plus TNF-α-treated cells. A micro (miR) array screen performed on RNA isolated from Wnt3a plus TNF-α treated and control cells demonstrated 13-fold up-regulation of miR203 by TNF-α; while in silico analyses indicated potential miR203 regulation of LOX mRNA. Analyses employing a miR203 mimic, and a reporter assay for active miR203, both demonstrated the functionality of TNF-α induced miR203 to target LOX mRNA.
Conclusion:
Wnt3a up-regulates LOX at the transcriptional level in C3H10T1/2 cells. TNF-α post-transcriptionally down-regulates LOX mediated by miR203. TNF-α may contribute to diabetic osteopenia by targeting LOX in mesenchymal stem cells, thereby resulting in deficient collagen cross-linking and osteopenia.