Native Human Osteocalcin Protein

Osteoporosis in advanced cholestatic and end-stage liver disease is related to low bone formation. Previous studies have demonstrated the deleterious consequences of lithocholic acid (LCA) and bilirubin on osteoblastic cells. These effects are partially or completely neutralized by ursodeoxycholic acid (UDCA). We have assessed the differential gene expression of osteoblastic cells under different culture conditions. The experiments were performed in human osteosarcoma cells (Saos-2) cultured with LCA (10 mu M), bilirubin (50 mu M) or UDCA (10 and 100 mu M) at 2 and 24 h. Expression of 87 genes related to bone metabolism and other signalling pathways were assessed by TaqMan micro fluidic cards. Several genes were up-regulated by LCA, most of them pro-apoptotic (BAX, BCL10, BCL2L13, BCL2L14), but also MGP (matrix Gla protein), BGLAP (osteocalcin), SPP1 (osteopontin) and CYP24A1, and down-regulated bone morphogenic protein genes (BMP3 and BMP4) and DKK1 (Dickkopf-related protein 1). Parallel effects were observed with bilirubin, which up-regulated apoptotic genes and CSF2 (colony-stimulating factor 2) and down-regulated antiapoptotic genes (BCL2 and BCL2L1), BMP3, BMP4 and RUNX2. UDCA 100 mu M had specific consequences since differential expression was observed, up-regulating BMP2, BMP4, BMP7, CALCR (calcitonin receptor), SPOCK3 (osteonectin), BGLAP (osteocalcin) and SPP1 (osteopontin), and down-regulating pro-apoptotic genes. Furthermore, most of the differential expression changes induced by both LCA and bilirubin were partially or completely neutralized by UDCA. Conclusion: Our observations reveal novel target genes, whose regulation by retained substances of cholestasis may provide additional insights into the pathogenesis of osteoporosis in cholestatic and end-stage liver diseases.

In general, methylation of the promoter regions is inversely correlated with gene expression. The transitional CpG area between the promoter-associated CpG islands and the nearby retroelements is often methylated in a tissue-specific manner. This study analyzed the relationship between gene expression and the methylation of the transitional CpGs in two human stromal cells derived from the bone marrow (BMSC) and adipose tissue (ATSC), both of which have a multi lineage differentiation potential. The transitional CpGs of the osteoblast-specific (RUNX2 and BGLAP), adipocyte-specific (PPAR gamma 2), housekeeping (CDKN2A and MLH1), and mesenchyme-unrelated (RUNX3) genes were examined by methylation-specific PCR. The expression of each gene was measured using reverse-transcription PCR analysis. The RUNX2, BGLAP, and CDKN2A genes in the BMSC, and the PPAR gamma 2 gene in the ATSC exhibited hypomethylation of the transitional CpGs along with the strong expression. The CpG island of RUNX3 gene not expressed in both BMSC and ATSC was hypermethylated. Transitional hypomethylation of the MLH1 gene was accompanied by the higher expression in the BMSC than in the ATSC. The weakly methylated CpGs of the PPAR gamma 2 gene in the BMSC became hypomethylated along with the strong expression during the osteoblastic differentiation. There were no notable changes in the transitional methylation and expression of the genes other than PPAR gamma 2 after the differentiation. Therefore, the transitional methylation and gene expression established in mesenchymal cells tend to be consistently preserved under the induction of differentiation. Weak transitional methylation of the PPAR gamma 2 gene in the BMSC suggests a methylation dependent mechanism underlying the adiopogenesis of bone marrow. J. Cell. Biochem. 102: 224-239, 2007. (C) 2007 Wiley-Liss, Inc.