Background: The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis).

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6β-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6β-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6β-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole.

Platelet activation by newly exposed basement membrane collagen is a key initial step in both hemostasis and thrombosis (1). The complex of glycoprotein VI (GPVI) 2 and the Fc receptor ␥-chain is primarily responsible for activation of platelets by collagen (2). This receptor acts via a PLC␥2-dependent pathway, leading to generation of autocoids that recruit additional platelets to the site of injury. Signaling downstream of the GPVI/Fc receptor ␥ chain is similar to signaling initiated by activation of immune receptors on T-and B-lymphocytes. The Fc receptor ␥-chain contains an immunoreceptor tyrosine activation motif. Binding of collagen to the receptor is thought to cluster the receptors, which initiates the phosphorylation of the immunoreceptor tyrosine activation motif tyrosines by an Src family kinase. Similar to signaling through other immune receptors, Syk tyrosine kinase binds to the phosphorylated immunoreceptor tyrosine activation motif to start a cascade of phosphorylations and protein interactions resulting in the activation of PLC␥2.Hematopoietic cells express two members of the Cbl family of E3 ligases namely, c-Cbl and Cbl-b (3-5). The Cbl family proteins have been shown to be key regulators of intracellular signaling in immune cells (6, 7). As E3 ligases, Cbl proteins can catalyze the transfer of ubiquitin molecules to their substrates. A primary role for ubiquitylation is targeting the substrates of E3 ligases to the proteasome, where the substrates are proteolytically degraded. Because of this activity, Cbl family members have come to be considered as negative regulators of signaling. In platelets, c-Cbl promotes ubiquitylation of Syk and appears to be a negative regulator of platelet activation. Murine platelets deficient in c-Cbl show enhanced platelet aggregation in response to convulxin, a GPVI agonist (8, 9). Furthermore, we have shown that Syk is not ubiquitylated in c-Cbl Ϫ/Ϫ platelets, in contrast to normal platelets (9).The presence of Cbl-b in platelets and any role it might have in signaling have not been investigated. Because Cbl-b is also an E3 ligase, one might expect that it would also be a negative regulator of cell function. However, several reports have indicated that Cbl-b may play a positive role in both T cell and B cell signaling. Cbl-b interacts with Zap-70, leading to activation of T-cell-specific transcription factor NF-AT (10). Cbl-b Ϫ/Ϫ DT40 B cells display reduced PLC␥2 activation and Ca 2ϩ mobilization upon B-cell receptor stimulation, and the overexpression of Cbl-b results in an enhanced agonist-dependent Ca 2ϩ mobilization (11). These results may be explained by another function of the Cbl molecules, namely that they have several domains in addition to those responsible for the E3 ligase activity, which, acting as a scaffold, can bind to a variety of signaling molecules, bringing them into close proximity (6,12,13).Given the importance of Cbl-b to regulation of signaling in lymphocytes, we have investigated whether Cbl-b regulates platelet activation downstream ...

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). IntroductionPlatelet activation is pivotal in the arrest of bleeding after vessel injury. Several pathways originating from G-protein-coupled receptors and integrins located on the platelet membrane contribute to platelet activation. For example, injury to the endothelium of a blood vessel leads to the release of tissue factor and exposure of subendothelial collagen, which initiates platelet activation. Collagen binds to platelets directly via the ␣2␤1 integrin and the glycoprotein VI (GPVI) receptor or indirectly through von Willebrand factor and GPIb/V/IX complex. [1][2][3][4][5] The binding of collagen to the GPVI receptor results in an intracellular signaling cascade that leads to platelet activation. 6,7 The GPVI receptor, a member of the immunoglobulin (Ig) superfamily, is coexpressed with Fc receptor ␥ (FcR␥) chain on platelets and serves as a single functional unit. 8 After the binding of collagen to GPVI, the FcR␥ chain is phosphorylated by Src kinases on the tyrosine residues of its immunoreceptor tyrosine-based activation motif (ITAM), 9,10 which, in turn, promotes the association of Syk kinase; consequently, Syk undergoes autophosphorylation. Activated Syk subsequently phosphorylates and activates phospholipase C␥2 (PLC␥2) through a cascade of signaling molecules involving LAT (linker for T-cell activation), phosphatidylinositol 3-kinase (PI3K), and Bruton tyrosine kinase. Activation of PLC␥2 leads to the rise in intracellular calcium and protein kinase C (PKC) activation, which results in secretion of dense granules, generation of thromboxane, and activation of the ␣IIb␤3 integrin, which leads to aggregation. 11 Activation of B and T lymphocytes leads to phosphorylation of a protein exclusively expressed in cells of hematopoietic lineage that is termed hematopoietic lineage cell-specific protein 1 (HS1). 12 HS1 is a 486-amino acid-long 75-kDa hydrophilic protein. 12 It is predominantly located in cytoplasm but after activation through tyrosine phosphorylation at residues 397, 378, and 222 13,14 translocates to plasma membrane. The protein also contains a HAX1 (HS-associated protein X-1)-binding site, an Src homology 3 (SH3) domain, a proline-rich region, and 3 additional phosphorylation sites. 14,15 These sites are sequentially phosphorylated after Band T-cell receptor cross-linking. After the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM), the nonreceptor tyrosine kinase Syk is phosphorylated and activated. Phosphorylated Syk binds to HS1, which results in tyrosine phosphorylation of HS1 at residues 397 and 378. Syk then dissociates from HS1, which allows Src-family kinases to bind to HS1 via its SH2 or SH3 domain. Src subsequently phosphorylates HS1 at tyrosine residue 222, which results in activation of the HS1 protein. Functionally, the HS1 protein has been shown to be involved in proliferation and apoptosis downstream of B-and T-cell receptor activation. 14...