p73 regulates basal and starvation-induced liver metabolism in vivo

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-benzylated L-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

Sulfate assimilation in higher plants. Characterization of a stable intermediate in the adenosine 5′-phosphosulfate reductase reaction

The enzyme catalysing the reduction of adenosine 5′-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

Cytochrome P450 4F isoforms in different species: Identification, gene regulation and putative roles in inflammation

The cytochrome P450 4F subfamily comprises a group of enzymes that metabolize derivatives of arachidonic acid such as prostaglandins, lipoxins leukotrienes and hydroxyeicosatetraenoic acids, which are important mediators involved in the inflammatory response. Therefore, we speculate that CYP4Fs might be able to modulate the extent of the inflammation by controlling of the tissue levels of these inflammatory mediators, especially, leukotriene B4. One way to provide support for this hypothesis is to test whether the expression of CYP4Fs changes under inflammatory conditions, since these changes are required to adjust the levels of inflammatory mediators. ^ A lipopolysacchride (LPS) induced rat inflammation model was used to analyze the expressions of rat CYP4F4 and CYP4F5 in liver and kidney. LPS administration did not change the constitutive expression level of CYP4F4 and CYP4F5. In liver, the expressions of CYP4F4 and CYP4F5 decreased to 50–60% of the untreated level. The same effect of LPS on CYP4F4 and CYP4F5 expression can be mimicked in hepatocyte primary cultures treated with LPS, indicating a direct of effect of LPS on hepatocytes. LPS treatment also decreased the activity of liver microsomes towards chlorpromazine, however, antibody inhibition study revealed that liver CYP4Fs are not the only players in metabolizing chlorpromazine. To study further the underlying mechanism, CYP4F5 gene was isolated, characterized, and the promoter region was defined. ^ Accumulating evidence showed that peroxisome proliferator-activated receptors (PPARs) play an active role in inflammation. To investigate the possible role of PPARα in regulating CYP4F expression by inflammation or by clofibrate treatment, the expressions of two new mouse 4F isoforms were analyzed in PPARα knockout mice upon LPS or clofibrate challenge. A novel induction of CYP4F15 by LPS and clofibrate was observed in kidney, and this effect is totally dependent on the presence of PPARα. Renal CYP4F16 expression was not affected by LPS or clofibrate in both (+/+) and (−/−) mice. In contrast, hepatic expressions of CYP4F15 and CYP4F16 were reduced significantly in (+/+) mice, but much less in (−/−) mice, suggesting that PPARα is partially responsible for this down-regulation. Clofibrate treatment reduced the expression of CYP4F16 in liver, but has no effect on CYP4F15 and PPARα does not have a role in hepatic CYP4F expression regulated by clofibrate. In general, CYP4Fs are regulated in an isoform-, tissue- and species-specific manner. ^ A human CYP4F isoform, CYP4F11, was isolated. The genomic structure was also solved by using database mining and bioinformatics tools. Localization of CYP4F11 to chromosome 19, 16 kb upstream of CYP4F2, suggests that human CYP4F genes may form a cluster on chromosome 19. This novel human 4F is highly expressed in liver, as well as in kidney, heart and skeletal muscle. Further study of the activity and gene regulation on CYP4F11 will provide us more insights into the physiological functions of CYP4F subfamily. ^

Use of cardiac glycosides for treatment of cancer: Determinants of cancer cell sensitivity

Cardiac glycoside compounds have traditionally been used to treat congestive heart failure. Recently, reports have suggested that cardiac glycosides may also be useful for treatment of malignant disease. Our research with oleandrin, a cardiac glycoside component of Nerium oleander, has shown it to be a potent inducer of human but not murine tumor cell apoptosis. Determinants of tumor sensitivity to cardiac glycosides were therefore studied in order to understand the species selective cytotoxic effects as well as explore differential sensitivity amongst a variety of human tumor cell lines. ^ An initial model system involved a comparison of human (BRO) to murine (B16) melanoma cells. Human BRO cells were found to express both the sensitive α3 as well as the less sensitive α1 isoform subunits of Na+,K +-ATPase while mouse B16 cells expressed only the α1 isoform. Drug uptake and inhibition of Na+,K+-ATPase activity were also different between BRO and B16 cells. Partially purified human Na+,K+-ATPase enzyme was inhibited by cardiac glycosides at a concentration that was 1000-fold less than that required to inhibit mouse B16 enzyme to the same extent. In addition, uptake of oleandrin and ouabain was 3–4 fold greater in human than murine cells. These data indicate that differential expression of Na+,K+-ATPase isoform composition in BRO and B16 cells as well as drug uptake and total enzyme activity may all be important determinants of tumor cell sensitivity to cardiac glycosides. ^ In a second model system, two in vitro cell culture model systems were investigated. The first consisted of HFU251 (low expression of Na+,K+-ATPase) and U251 (high Na+ ,K+-ATPase expression) cell lines. Also investigated were human BRO cells that had undergone stable transfection with the α1 subunit resulting in an increase in total Na+,K+-ATPase expression. Data derived from these model systems have indicated that increased expression of Na+,K+-ATPase is associated with an increased resistance to cardiac glycosides. Over-expression of Na +,K+-ATPase in tumor cells resulted in an increase of total Na+,K+-ATPase activity and, in turn, a decreased inhibition of Na+,K+-ATPase activity by cardiac glycosides. However, of interest was the observation that increased enzyme expression was also associated with an elevated basal level of glutathione (GSH) within cells. Both increased Na+,K+-ATPase activity and elevated GSH content appear to contribute to a delayed as well as diminished release of cytochrome c and caspase activation. In addition, we have noted an increased colony forming ability in cells with a high level of Na+,K+-ATPase expression. This suggests that Na+,K+-ATPase is actively involved in tumor cell growth and survival. ^

CCTeta: A novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of 3′,5 ′-cyclic guanosine monophosphate (cGMP) from 5′ -guanosine triphosphate (GTP). In this thesis, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β1 isoform of sGC. Using the yeast-two-hybrid system, CCTη was found to interact with the N-terminal portion of β1 subunit of sGC. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast-two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 33% inhibition of sodium nitroprusside (SNP)-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on the activity of αβCys105 sGC a constitutively active mutant that lacks a heme group. Of note is the fact that the full-length CCTη-expressing bacterial lysate inhibited the activity of sGC-expressing Sf9 lysate by 48% compared with GST alone. This indicates that the amino terminal 94 amino acids of CCTη are important to the inhibition of sGC activity. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with SNP. The fact that the inhibition of sGC was more pronounced with bacterial lysate expressing CCTη versus the purified CCTη implies that some factor in the bacterial lysate enhances the inhibitory effect of CCTη. Because the level of inhibition seen in bacterial lysate and in vivo experiments is similar, might imply that the factor that aids in CCTη effect on sGC is conserved. Together, these data suggest that CCTη is a novel type of sGC inhibitor that inhibits sGC by modifying the binding of NO to the heme group or the subsequent conformational changes induced by NO binding. ^

Correction of aberrant FGFR1 RNA splicing through blocking intronic regulatory elements

Analysis of the human genome has revealed that more than 74% of human genes undergo alternative RNA splicing. Aberrations in alternative RNA splicing have been associated with several human disorders, including cancer. ^ We studied the aberrant expression of alternative RNA splicing isoforms of the Fibroblast Growth Factor Receptor 1 (FGFR1) gene in a human glioblastoma cancer model. Normal glial cells express the FGFR1α, which contains three extracellular domains. In tumors the most abundant isoform is the FGFR1β, which lacks the first extracellular domain due to the skipping of a single exon, termed alpha. The skipping of the α-exon is regulated by two intronic silencing sequences within the precursor mRNA. Since we observed no mutations on these elements in tumor cells, we hypothesized that the over-expression of regulatory proteins that recognize these sequences is responsible for the aberrant expression of splicing isoforms. Hence, we blocked the formation of protein complexes on the ISS using antisense RNA oligonucleotides in vitro. We also evaluated the impact of the ISS antisense oligonucleotides on the endogenous FGFR1 splicing, in a glioblastoma cell model. By targeting intronic regulatory elements we were able to increase the level of alpha exon inclusion up to 90% in glioblastoma cells. The effect was dose dependent, sequence specific and reproducible in glioblastoma and other cancer cells, which also exhibit an alpha exon skipping phenotype. Targeting FGFR1 endogenous ISS1 and ISS2 sequences did not have an additive or synergistic effect, which suggest a regulatory splicing mechanism that requires the interaction of complexes formed on these elements. An increase in the levels of the FGFR1α isoform resulted in a reduction in cell invasiveness. Also, a significant increase in the levels of caspase 3/7 activities, which is indicative of an elevation in apoptosis levels, suggests that expression of FGFR1β might be relevant for tumor survival. These studies demonstrate that it is possible to prevent aberrant expression of exon skipping events through the targeting of intronic regulatory elements, providing an important new therapeutic tool for the correction of human disease caused by alternative RNA splicing. ^

Molecular mechanisms of signal transducer and activator of transcription (STAT) protein function in hematopoiesis

Hematopoietic growth factors play important roles in regulating blood cell growth and development in vivo. In this work, we investigated the signaling mechanisms of two growth factors with clinical significance, erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF). Epo is essential for the survival, proliferation and differentiation of red blood cell progenitors, while G-CSF plays an important role in controlling mature neutrophil production. To identify which amino acid(s) and/or motif in EpoR is responsible for cell survival, wild type or mutant EpoR isoforms were transfected into the growth factor-dependent 32D cell line. Proliferation and apoptosis assays demonstrated that an EpoR isoform that lacks intracellular tyrosine residues and is truncated after 321 amino acids in the cytoplasmic tail (EpoR 1-321) mediates Epo-dependent cell survival. Furthermore, in absence of fetal calf serum (FCS), Epo signaling through wild type or mutant receptors supported anti-apoptosis, but not proliferation during 72 hours in response to Epo. To investigate the signaling pathway by which EpoR regulates cell survival, a dominant negative Stat5b (dnStat5b) isoform was generated and coexpressed with EpoR in stable cell lines. Expression of dnStat5b causes a significant induction of apoptosis in the presence of Epo in cells expressing EpoR 1-321, indicating that Stat5 is essential for survival signaling through tyrosine independent sequences in the EpoR. In a second project to investigate G-CSF signaling, we studied mechanisms by which G-CSF regulates the expression of PU.1, an important transcription factor in myeloid and B cell development. We demonstrated, by immunoblot and real time RT-PCR, that PU.1 is induced by G-CSF ex vivo as well as in vivo. To test whether G-CSF signaling through Stat3 is required for PU.1 regulation, the upstream region of the PU.1 gene was analyzed for potential Stat3 binding motifs. Four potential sites were identified; chromatin immunoprecipitations demonstrated that G-CSF activated Stat3 binds to 3 of the 4 binding motifs. In addition, PU.1 induction by G-CSF was completely abrogated in bone marrow from hematopoietic conditional Stat3 knockout mice. These results indicate an important role for Stat3 in G-CSF-dependent PU.1 gene regulation. Collectively, our works demonstrate that Stat protein play important and diverse roles in hematopoietic growth factor signaling. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Female-specific regulation of cytochrome P450 3As and their response to xenobiotic stress

Cytochrome P450 3As (CYP3As) are phase I enzymes responsible for metabolizing more than 50% of clinical drugs. Recent studies have revealed that expression of CYP3As is two-fold higher in women than in men leading to a faster metabolic clearance of therapeutic drugs in women. In this study, we analyzed the female specific rat CYP3A isoform, CYP3A9. We evaluated the effects of progesterone and estrogen on CYP3A9 regulation and showed a distinct role for estrogen in mediating female dominance of CYP3A9. We also observed changes in CYP3A9 expression at various stages of pregnancy which correlates well with varying physiological estradiol concentrations. In addition, by the in vitro data shows that estradiol mediated induction can be abrogated with estrogen receptor antagonist ICI182,780. We also identified three novel murine CYP3A isoforms CYP3A13, CYP3A41 and CYP3A44 and characterized their genomic structures and expression profiles. CYP3A41 and CYP3A44 show female specific expression but surprisingly this female dominance is not mediated via estrogen. Control male mice did not exhibit any CYP3A41 mRNA levels but showed minimal levels of CYP3A44. In order to gain insights into the governance ofαthe female specific genes, the hepatic regulation of CYP3A41 and CYP3A44 by the xeno-sensors PXR and CAR was examined. In female mice, pregnenolone-16α-carboxynitrile, suppressed CYP3A41 and CYP3A44 mRNA levels in PXR−/− background whereas dexamethasone-dependent suppression of CYP3A41 was mediated by PXR. In addition, phenobarbital challenge in PXR−/− revealed up-regulation of both CYP3A44, CYP3A41 levels only in males. No role for CAR was seen in the regulation of either CYP3A41 or CYP3A44 gene expression in female mice. Interestingly, PXR and CAR ligands induced male CYP3A44 levels in a receptor dependent fashion. This increase of CYP3A44 transcript in male mice is in contrast to the response seen in female mice, which clearly indicates an additional layer of regulation. Our findings suggest that gender plays a strategic role in directing the CAR/PXR mediated effects of CYP3A44/CYP3A41. This implies that differential regulation of female specific CYP3A isoforms may be the key to explain some of the gender differences observed in clearance of certain therapeutics like antidepressants and analgesics. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^