Three patterns of cytochrome P450 gene expression during liver maturation in mice

The neonatal period of liver development is an often overlooked phase of development. For instance, ontogeny of xenobiotic-metabolizing enzymes can markedly affect biotransformation as the liver matures. To systematically examine the ontogenic gene expression patterns of cytochrome P450 genes (P450) in mice, the gene expression profiles of 19 xenobiotic-metabolizing P450 in Cyp1 to 4 families were determined. The mRNA levels in C57BL/6 mouse livers were quantified using branched DNA technology at the following ages: gestational day 17 (2 days before birth) and postnatal days 0, 1, 3, 5, 10, 15, 20, 30, and 45. Among the 13 P450 genes expressed in mouse livers, three distinct ontogenic expression patterns were identified by cluster analysis. Genes in group 1 (Cyp3a16 as well as 3a41b in male) were expressed in the perinatal period, but they were essentially nondetectable by 30 days of age. Genes in group 2 (Cyp2e1, 3a11, and 4a10 as well as 3a41b in female) quickly increased after birth and reached maximal expression levels by day 5. Genes in group 3 (Cyp1a2, 2a4, 2b10, 2c29, 2d22, 2f2, 3a13, and 3a25) were expressed at low levels until days 10 to 15, but they markedly increased at day 20 to a high and stable level. In conclusion, the developmental expression of P450 in mouse liver can be divided into three patterns, suggesting that different mechanisms are responsible for the expression of P450 during liver maturation.     

Cell-specific effects of TNF-α and IL-1β on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification

Tumor necrosis factor (TNF)-α and interleukin (IL)-1β stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-α therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-α and IL-1β on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-α and IL-1β for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-α and IL-1β significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-α stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor γ (PPARγ), which is inhibited by TNF-α. Indeed, in human chondrocytes and VSMCs, the PPARγ inhibitor GW-9662 displayed the same opposite effects as TNF-α on TNAP expression. In conclusion, whereas TNF-α and IL-1β stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPARγ as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification.     

Structure-activity relationships of alpha-ketooxazole inhibitors of fatty acid amide hydrolase

A systematic study of the structure-activity relationships of 2b (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed targeting the C2 acyl side chain. A series of aryl replacements or substituents for the terminal phenyl group provided effective inhibitors (e.g., 5c, aryl = 1-napthyl, Ki = 2.6 nM), with 5hh (aryl = 3-ClPh, Ki = 900 pM) being 5-fold more potent than 2b. Conformationally restricted C2 side chains were examined, and many provided exceptionally potent inhibitors, of which 11j (ethylbiphenyl side chain) was established to be a 750 pM inhibitor. A systematic series of heteroatoms (O, NMe, S), electron-withdrawing groups (SO, SO2), and amides positioned within and hydroxyl substitutions on the linking side chain were investigated, which typically led to a loss in potency. The most tolerant positions provided effective inhibitors (12p, 6-position S, Ki = 3 nM, or 13d, 2-position OH, Ki = 8 nM) comparable in potency to 2b. Proteome-wide screening of selected inhibitors from the systematic series of >100 candidates prepared revealed that they are selective for FAAH over all other mammalian serine proteases.