Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype.

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.

Involvement of cyclooxygenase-2 in the potentiation of allyl alcohol-induced liver injury by bacterial lipopolysaccharide.

Bacterial endotoxin (lipopolysaccharide; LPS) augments the hepatotoxicity of a number of xenobiotics including allyl alcohol. The mechanism for this effect is known to involve the inflammatory response elicited by LPS. Upregulation of cyclooxygenase-2 (COX-2) and production of eicosanoids are important aspects of inflammation, therefore studies were undertaken to investigate the role of COX-2 in LPS-induced enhancement of liver injury from allyl alcohol. Rats were pretreated (iv) with a noninjurious dose of LPS or sterile saline vehicle and 2 h later were treated (ip) with a noninjurious dose of allyl alcohol or saline vehicle. COX-2 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and liver injury was assessed from activities in serum of alanine and aspartate aminotransferases (ALT and AST, respectively) and from histology. Liver injury was observed only in rats cotreated with LPS and allyl alcohol. Serum ALT activity was increased by 4 h after administration of LPS and continued to increase through 8 h. COX-2 mRNA was detectable at low levels in livers from rats receiving only the vehicles at any time up to 8 h. Expression of COX-2 mRNA was increased by 30 min after administration of LPS and remained elevated through 6 h. Allyl alcohol treatment alone caused an increase in COX-2 mRNA at 4 h (2 h after allyl alcohol) that lasted less than 2 h. In livers from rats cotreated with LPS and allyl alcohol, levels of COX-2 mRNA were greater than levels seen with either LPS or allyl alcohol alone. The increased expression of COX-2 mRNA was accompanied by an increase in the concentration of prostaglandin (PG) D(2) in plasma. Plasma PGD(2) concentration was increased to a greater extent in rats treated with LPS plus allyl alcohol compared to allyl alcohol or LPS alone. Pretreatment with the COX-2 selective inhibitor, NS-398, abolished the increase in plasma PGD(2) and reduced the increase in ALT and AST activities observed in rats cotreated with LPS and allyl alcohol. NS-398 did not affect liver injury from allyl alcohol alone administered at a larger, hepatotoxic dose. In addition, ibuprofen, a nonselective inhibitor of cyclooxygenases, did not protect against liver injury from LPS plus allyl alcohol. In isolated hepatocytes PGD(2), but not PGE(2), reduced the concentration of allyl alcohol required to cause half-maximal cytotoxicity. These results suggest that products of COX-2 play a role in the augmentation of allyl alcohol-induced liver injury by LPS.

Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline.

Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide [LPS]) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation. Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication. Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats. In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity. Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination. Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury. Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h. The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively. In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone. A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity. In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury. Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells. These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins.