Bile acids and bilirubin effects on osteoblastic gene profile. Implications in the pathogenesis of osteoporosis in liver diseases.

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 µM), bilirubin (50 µM) or UDCA (10 and 100 µM) 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 µM 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.

Adapting through glacial cycles: insights from a long-lived tree (Taxus baccata).

Despite the large body of research devoted to understanding the role of Quaternary glacial cycles in the genetic divergence of European trees, the differential contribution of geographic isolation and/or environmental adaptation in creating population genetic divergence remains unexplored. In this study, we used a long-lived tree (Taxus baccata) as a model species to investigate the impact of Quaternary climatic changes on genetic diversity via neutral (isolation-by-distance) and selective (isolation-by-adaptation) processes. We applied approximate Bayesian computation to genetic data to infer its demographic history, and combined this information with past and present climatic data to assess the role of environment and geography in the observed patterns of genetic structure. We found evidence that yew colonized Europe from the East, and that European samples diverged into two groups (Western, Eastern) at the beginning of the Quaternary glaciations, c. 2.2 Myr before present. Apart from the expected effects of geographical isolation during glacials, we discovered a significant role of environmental adaptation during interglacials at the origin of genetic divergence between both groups. This process may be common in other organisms, providing new research lines to explore the effect of Quaternary climatic factors on present-day patterns of genetic diversity.

Ursodeoxycholic acid decreases bilirubin-induced osteoblast apoptosis.

BACKGROUND: Low bone turnover osteoporosis is common in cholestatic diseases. Ursodeoxycholic acid (UDCA) counteracts the damaging effects of bilirubin or lithocholic acid (LCA) on osteoblast viability, proliferation and mineralisation. UDCA is anti-apoptotic in various cell lines, but this effect in bone cells is unknown. Therefore, the consequences of bilirubin and LCA on apoptosis, and whether UDCA has anti-apoptotic effects have been assessed on osteoblasts. MATERIALS AND METHODS: Human osteoblasts (hOB) and osteosarcoma cell line (Saos-2) were treated with camptothecin as a pro-apoptotic agent, and UDCA, LCA and bilirubin. Apoptosis was determined by DNA fragmentation, flow cytometry, caspase-3 activity and expression of pro-apoptotic (Bcl-2-associated X protein BAX) and anti-apoptotic (BCL2 and BCL2-like 1 protein, BCL2L) genes. RESULTS: Both LCA (10 µM) and bilirubin (50 µM) induced apoptosis as indicated by DNA fragmentation (4·7- and 3·7-fold, respectively, P < 0·001), caspase-3 activity and flow cytometry in Saos-2 and hOB. UDCA (10 µM) reduced the apoptotic effects of camptothecin (0·5 µM) by 61%, (P < 0·001) and counteracted the apoptotic effects of LCA and bilirubin determined by DNA fragmentation (56% and 60%, respectively, P < 0·001), cytometry and caspase-3 activity in Saos-2, with lower effects in hOB. UDCA (10 µM) downregulated BAX (75%), upregulated BCL2L (10-fold, P < 0·01) genes, and neutralised BAX upregulation (P < 0·01) and BCL2L downregulation (P < 0·01) induced by LCA and bilirubin. CONCLUSIONS: Bilirubin and LCA induce apoptosis in osteoblastic cells. UDCA counteracts the apoptotic consequences of these two substances, and therefore, it may have further beneficial effects on the decreased bone formation in the cholestasis.

Ursodeoxycholic acid increases differentiation and mineralization and neutralizes the damaging effects of bilirubin on osteoblastic cells.

BACKGROUND: Osteoporosis resulting from decreased bone formation is a common complication in patients with chronic cholestasis. Lithocholic acid (LCA) and bilirubin may play a role in osteoporosis given that both substances have detrimental effects on survival of human osteoblasts, the cells involved in bone formation. AIMS: As ursodeoxycholic acid (UDCA) improves cholestasis, we have assessed if this bile acid may neutralize the harmful effects of LCA, bilirubin and sera from jaundiced patients on osteoblastic cells. METHODS: The experiments were performed in primary human osteoblasts and human osteosarcoma cell line (Saos-2) at different times and concentrations of UDCA, LCA, cholic acid (CA), bilirubin and sera from jaundiced patients to assess cell viability, differentiation and mineralization. RESULTS: UDCA significantly decreased cell survival at concentrations 10 times higher (1 mM) than that observed with LCA, whereas CA did not decrease osteoblast survival. UDCA (100 µM) neutralized the damaging effects of bilirubin (50 µM) and sera from jaundiced patients on survival. Moreover, UDCA (1 µM and 10 µM) increased osteoblast differentiation in cells treated with harmful concentrations of LCA or bilirubin. UDCA (100 µM) increased cell differentiation in osteoblasts cultured with a mix of serum from cholestatic patients by 23%. Furthermore, UDCA increased osteoblast mineralization by 35% and neutralized the negative consequences of 50 µM bilirubin. CONCLUSIONS: UDCA increases osteoblast differentiation and mineralization, and neutralizes the detrimental effects of lithocholic acid, bilirubin and sera from jaundiced patients on osteoblastic cells. Therefore, UDCA may exert a favourable effect on bone in patients which chronic cholestasis.

Effects of bilirubin and sera from jaundiced patients on osteoblasts: contribution to the development of osteoporosis in liver diseases.

UNLABELLED: Low bone formation is considered to be the main feature in osteoporosis associated with cholestatic and end-stage liver diseases, although the consequences of retained substances in chronic cholestasis on bone cells have scarcely been studied. Therefore, we analyzed the effects of bilirubin and serum from jaundiced patients on viability, differentiation, mineralization, and gene expression in the cells involved in bone formation. The experiments were performed in human primary osteoblasts and SAOS-2 human osteosarcoma cells. Unconjugated bilirubin or serum from jaundiced patients resulted in a dose-dependent decrease in osteoblast viability. Concentrations of bilirubin or jaundiced serum without effects on cell survival significantly diminished osteoblast differentiation. Mineralization was significantly reduced by exposure to 50 µM bilirubin at all time points (from -32% to -55%) and jaundiced sera resulted in a significant decrease on cell mineralization as well. Furthermore, bilirubin down-regulated RUNX2 (runt-related transcription factor 2) gene expression, a basic osteogenic factor involved in osteoblast differentiation, and serum from jaundiced patients significantly up-regulated the RANKL/OPG (receptor activator of nuclear factor-κB ligand/osteoprotegerin) gene expression ratio, a system closely involved in osteoblast-induced osteoclastogenesis. CONCLUSION: Besides decreased cell viability, unconjugated bilirubin and serum from jaundiced patients led to defective consequences on osteoblasts. Moreover, jaundiced serum up-regulates the system involved in osteoblast-induced osteoclastogenesis. These results support the deleterious consequences of increased bilirubin in advanced chronic cholestasis and in end-stage liver diseases, resulting in disturbed bone formation related to osteoblast dysfunction.

Genetic effects of chronic habitat fragmentation revisited: Strong genetic structure in a temperate tree, Taxus baccata (Taxaceae), with great dispersal capability.

Tree species are thought to be relatively resistant to habitat fragmentation because of their longevity and their aptitude for extensive gene flow, although recent empirical studies have reported negative genetic consequences, in particular after long-term habitat fragmentation in European temperate regions. Yet the response of each species to habitat loss may differ greatly depending on their biological attributes, in particular seed dispersal ability. In this study, we used demographic and molecular data to investigate the genetic consequences of chronic habitat fragmentation in remnant populations of Taxus baccata in the Montseny Mountains, northeast Spain. The age structure of populations revealed demographic bottlenecks and recruitment events associated with exploitation and management practices. We found a strong genetic structure, both at the landscape and within-population levels. We also detected high levels of inbreeding for a strictly outcrossing species. Chronic forest fragmentation resulting from long-term exploitation in the Montseny Mountains seems the most plausible explanation for the strong genetic structure observed. Our results support the view that, contrary to some predictions, tree species are not buffered from the adverse effects of habitat fragmentation, even in the case of species with a high dispersal potential.

Genetic structure and diversity in Ramonda myconi (Gesneriaceae): effects of historical climate change on a preglacial relict species.

The importance of the Mediterranean Basin as a long-term reservoir of biological diversity has been widely recognized, although much less effort has been devoted to understanding processes that allow species to persist in this area. Ramonda myconi (Gesneriaceae) is a Tertiary relict plant species restricted to the NE Iberian Peninsula. We used RAPD and chloroplast markers to assess the patterns of genetic structure in eight mountain regions covering almost the full species range, to identify the main historical processes that have shaped its current distribution and to infer the number and location of putative glacial refugia. While no cpDNA variation was detected, the species had relatively high levels of RAPD variation. Maximum levels of diversity were found within populations (71%), but there was also a significant differentiation between geographical regions (20%) and among populations within regions (9%). A spatial AMOVA identified three main groups of populations, corresponding to previously recognized centers of endemism and species richness. In addition, we found a marked geographical pattern of decreasing genetic diversity and increasing population differentiation from west to east. Our results support a complex phylogeographic scenario in the Iberian Peninsula of "refugia-within-refugia" and suggest that the higher diversity observed in western regions might be associated with prolonged and more stable climatic conditions in this area during the Quaternary.