23-25 On the other hand, the findings from this study also indicate that unconjugated bilirubin and sera from jaundiced patients induced significant changes on gene expression, even in osteoblasts treated for a short time (that is, 24 hours). Thus, exposure to bilirubin at 50 μM decreased cell differentiation, as demonstrated by a significant down-regulation of RUNX2, effects which were already observed at low concentrations of sera (2%). This result is in agreement with the decreased alkaline phosphatase activity.

The lack of effect of sera from jaundiced patients on Talazoparib clinical trial RUNX2 gene expression can be, to some degree, explained by various factors: (1) increased total bilirubin and lesser amount of unconjugated bilirubin; (2) the presence of molecules other than bilirubin in the media; and (3) the short time used in the experiments, taking into account that osteocalcin is a late marker for osteoblast differentiation. These concerns can also account for the lack of effect of bilirubin and sera from jaundiced patients on

osteocalcin gene expression, which was not significantly decreased in the experiments with primary human osteoblasts. This result is in contrast with other experiments performed in primary rat osteoblasts, indicating that Doxorubicin cell line very low bilirubin concentrations (3 and 30 μM) down-regulated the expression of osteocalcin and RUNX2 in osteoblasts cultured in osteogenic medium for 3 to 14 days.22 The OPG/RANKL system is the key regulator of osteoblast-induced osteoclastogenesis, because both osteoprotegerin and RANKL are primarily synthesized in osteoblasts but eventually act on osteoclasts, the cell line responsible for bone resorption. Thus, osteoblasts produce RANKL, a ligand for the receptor activator of nuclear factor-κB (RANK) on hematopoietic cells, which activates the differentiation of osteoclasts and maintains their function. Osteoblasts

also produce and secrete osteoprotegerin, a decoy receptor that can block RANKL/RANK not interactions.26 Even though the experiments were performed with a short period of incubation, jaundiced sera influenced the expression of RANKL and OPG. Serum from jaundiced patients was able to up-regulate RANKL and down-regulate OPG gene expression, thus resulting in a very significant increase in the RANKL/OPG gene expression ratio. These novel data derived from this study can be extrapolated to what is observed in clinical conditions in patients with chronic cholestatic diseases, in whom, besides decreased bone formation, a parallel increase in bone resorption has been described,4, 27 an event that has been attributed to secondary hyperparathyroidism resulting from overt or subtle deficiencies in calcium and vitamin D levels as a consequence of intestinal malabsorption.