No abstract

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Epoxy-and dihydroxy-eicosatrienoic acids (EETs and DHETs) are vasoactive cytochrome P450 metabolites of arachidonic acid. Interestingly, however, the mechanism(s) by which EETs/ DHETs mediate smooth muscle relaxation remains unclear. In contrast to previous reports, where dilation was purportedly large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) and/or transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel-mediated, 14,15-EET-induced vasodilation [reversal of contractile tone established with the thromboxane receptor (TP) agonist 15-hydroxy-11␣,9␣-(epoxymethano)prosta-5,13-dienoic acid (U-46619)] was unaltered in BK Ca and TRPV4 knockout mouse isolated aortae compared with wild-type controls, indicating a significant BK Ca /TRPV4-resistant mechanism. Whereas all EET and DHET regioisomers reversed U-46619 contraction in rat aortae and mouse mesenteric resistance arteries, these eicosanoids failed to alter phenylephrine-induced contraction, suggesting that they mediated dilation via a "TP-selective" mechanism. Competitive TP antagonism was also observed in nonvascular tissue, including rat fundus and tertiary bronchus, indicating that the effect is not specific to blood vessels. Such effects were TP-selective because 14,15-EET failed to inhibit "non-TP" prostanoid receptor-mediated function in multiple cell/ tissue-based assays (K b Ͼ 10 M). In accordance, 14,15-EET inhibited specific [ 3 H]7-(3-((2-((phenylamino)carbonyl)hydrazino)-methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ-29548) binding to human recombinant TP receptor, with a K i value of 3.2 M, and it showed weaker affinity for non-TP prostanoid receptors, including DP, FP, EP 1-4 , and IP receptors (K i values of 6.1, 5.3, 42.6, 19.7, 13.2, 20.2, and Ͼ25 M, respectively) and no appreciable affinity (K i values Ͼ10 M) for a diverse array of pharmacologically distinct receptors, including the leukotriene receptors Cys-LT 1/2 and BLT 1 . As such, EETs/ DHETs represent a unique class of "endogenous" G proteincoupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Thus, EETs/DHETs represent novel autoregulatory agents, directly modulating the actions of cyclooxygenase-derived eicosanoids following arachidonic acid mobilization.Upon release from cell membranes, arachidonic acid can be converted to a range of eicosanoids by three principal classes of enzymes: cyclooxygenases (COX), lipoxygenases, and cytochrome P450 monooxygenases. The P450 epoxygenases can introduce an epoxide to any of the four double bonds (5,6, 8,9, 11,12, and 14,15) of arachidonic acid, resulting in the generation of four distinct EET regioisomers (Capdevila et al., 1990). Each EET can be further metabolized to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH), resulting in the generation of four corresponding DHET regioisomers.Since their discovery more than 25 years ago (Capdevila et Article, publication date, and citation information can be found at

The protein tyrosine phosphatase SHP-1 is critical for controlling cytokine signaling through the Jak-Stat pathway and, consequently, for controlling inflammatory cellular immune responses dependent on these cytokines. However, the role of SHP-1 in regulating proinflammatory signaling may be incompletely understood, and it may control other distinct inflammatory agents. The present study analyzed the ability of tumor necrosis factor-alpha (TNF-alpha), double-stranded RNA, and interferon-gamma (IFN-gamma) to induce the transcription factor NF-kappaB in astrocytes expressing or lacking SHP-1. On exposure to the inducers, NF-kappaB was markedly increased in astrocytes obtained from motheaten mice lacking SHP-1 compared with normal littermate cells expressing SHP-1, consisted of p50 and p65 subunits, and was induced in a protein synthesis-independent manner. The increased nuclear NF-kappaB expression coincided with elevated loss of the cytoplasmic inhibitor IkappaB alpha in motheaten mouse cells. Enhanced NF-kappaB expression in motheaten mouse cells correlated with increased expression of genes with functional kappaB sites, including IFN regulatory factor-1 (IRF-1) and inducible nitric oxide synthase (iNOS) genes. MHC class I molecules were also increased in motheaten cells, consistent with the increased expression of IRF-1. Together, the data indicate an increased sensitivity of cells lacking SHP-1 to various inducers of NF-kappaB. Therefore, the regulation of not only Stats but also of NF-kappaB by SHP-1 may be important in controlling events promoted by proinflammatory agents in vivo that are especially apparent in multiple tissues of motheaten mice. This study suggests an additional role for SHP-1 in controlling specific and nonspecific immune responses where induction of NF-kappaB is involved.

Previous studies in this laboratory have shown that the SH‐2 domain‐containing protein tyrosine phosphatase SHP‐1 is expressed in CNS glia and functions to modulate cytokine activities in these cells. The present study demonstrates that SHP‐1 is expressed within multiple regions of the CNS in vivo, especially in white matter. Interestingly, we show that mice genetically lacking in SHP‐1 (motheaten mice) in the CNS displayed dysmyelination. We therefore examined the expression of SHP‐1 in the myelin‐forming oligodendrocytes. Oligodendrocytes present in either mixed glial cultures or pure cultures expressed high levels of SHP‐1 in the cytoplasm of cell bodies and processes. Oligodendrocytes isolated from motheaten mice did not express SHP‐1. To test possible functions for SHP‐1 in oligodendrocytes in controlling cytokine signaling, we compared the responsiveness of oligodendrocytes isolated from either motheaten or normal littermate mice with IL‐6. IL‐6 induced higher levels of STAT3 phosphorylation and STAT3‐responsive c‐fos gene expression in pure oligodendrocyte cultures of motheaten compared with normal littermate mice. These studies demonstrate that oligodendrocytes express SHP‐1 and that SHP‐1 functions to control IL‐6 signaling. SHP‐1 may therefore be a critical regulator of oligodendrocyte differentiation in response to IL‐6 family cytokines. Further, these findings may relate to dysmyelination in mice lacking SHP‐1. GLIA 29:376–385, 2000. © 2000 Wiley‐Liss, Inc.