Pharmacological Characterization of a Potent Inhibitor of Autotaxin in Animal Models of Inflammatory Bowel Disease and Multiple Sclerosis.

Autotaxin is a secreted enzyme that catalyzes the conversion of lysophosphatidyl choline into the bioactive lipid mediator lysophosphatidic acid (LPA). It is the primary enzyme responsible for LPA production in plasma. It is upregulated in inflammatory conditions and inhibition of autotaxin may have anti-inflammatory activity in a variety of inflammatory diseases. To determine the role of autotaxin and LPA in the pathophysiology of inflammatory disease states, we used a potent and orally bioavailable inhibitor of autotaxin that we have recently identified, and characterized it in mouse models of inflammation, inflammatory bowel disease (IBD), multiple sclerosis (MS), and visceral pain. Compound-1, a potent inhibitor of autotaxin with an IC50 of ∼2 nM, has good oral pharmacokinetic properties in mice and results in a substantial inhibition of plasma LPA that correlates with drug exposure levels. Treatment with the inhibitor resulted in significant anti-inflammatory and analgesic effects in the carrageenan-induced paw inflammation and acetic acid-induced visceral pain tests, respectively. Compound-1 also significantly inhibited disease activity score in the dextran sodium sulfate-induced model of IBD, and in the experimental autoimmune encephalomyelitis model of MS. In conclusion, the present study demonstrates the anti-inflammatory and analgesic properties of a novel inhibitor of autotaxin that may serve as a therapeutic option for IBD, MS, and pain associated with inflammatory states.

Human anti-prion antibodies block prion peptide fibril formation and neurotoxicity.

Prion diseases are a group of rare, fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K-resistant conformer, termed scrapie PrP (PrP(Sc)). Aggregates of PrP(Sc) deposited around neurons lead to neuropathological alterations. Currently, there is no effective treatment for these fatal illnesses; thus, the development of an effective therapy is a priority. PrP peptide-based ELISA assay methods were developed for detection and immunoaffinity chromatography capture was developed for purification of naturally occurring PrP peptide autoantibodies present in human CSF, individual donor serum, and commercial preparations of pooled intravenous immunoglobulin (IVIg). The ratio of anti-PrP autoantibodies (PrP-AA) to total IgG was ∼1:1200. The binding epitope of purified PrP-AA was mapped to an N-terminal region comprising the PrP amino acid sequence KTNMK. Purified PrP-AA potently blocked fibril formation by a toxic 21-amino acid fragment of the PrP peptide containing the amino acid alanine to valine substitution corresponding to position 117 of the full-length peptide (A117V). Furthermore, PrP-AA attenuated the neurotoxicity of PrP(A117V) and wild-type peptides in rat cerebellar granule neuron (CGN) cultures. In contrast, IgG preparations depleted of PrP-AA had little effect on PrP fibril formation or PrP neurotoxicity. The specificity of PrP-AA was demonstrated by immunoprecipitating PrP protein in brain tissues of transgenic mice expressing the human PrP(A117V) epitope and Sc237 hamster. Based on these intriguing findings, it is suggested that human PrP-AA may be useful for interfering with the pathogenic effects of pathogenic prion proteins and, thereby has the potential to be an effective means for preventing or attenuating human prion disease progression.

Estrogen promotes tumor progression in a genetically defined mouse model of lung adenocarcinoma.

Numerous epidemiological observations point to sex differences in lung cancer etiology and progression. The present study was aimed at understanding the bases of these sex differences. To test the effect of estradiol on tumor progression, we used a mouse model based on conditional Kras expression and concurrent deletion of Tp53 following inhalation of an adenoviral vector expressing Cre recombinase (AdeCre). Ovariectomized females and males were treated with estradiol via a continuous-release capsule. Tumor multiplicity, tumor volume, and histological grade were determined at 10 weeks after AdeCre administration. Cell proliferation was monitored by Ki67 immunohistochemistry at 4 and 10 weeks after AdeCre administration. At 10 weeks, female mice had more than twice the number of tumors evident on the surface of the lungs than male mice; ovariectomy eliminated this sex difference. The estrogen treatment significantly increased tumor number and volume in ovariectomized females and in males. Histological character of the tumors ranged from adenoma to adenocarcinoma. Ovary-intact females exhibited higher grade tumors than ovariectomized females or males. Progression to higher histological grade was stimulated by estrogen in male mice but not in ovariectomized females. At 10 weeks after AdeCre administration, tumor cell Ki67-labeling varied widely, precluding assessment of an estrogen effect; however, at 4 weeks, Ki67 labeling of lung parenchymal cells was increased 3.5-fold by estrogen. In conclusion, estrogen acts as a promoter for lung adenocarcinoma in a genetically defined lung cancer model; estrogen-induced cell proliferation in the oncogene-initiated cells is likely to play a role in this tumor promoter activity.

Striatin-3 gamma inhibits estrogen receptor activity by recruiting a protein phosphatase.

A splicing variant of rat striatin-3 (rSTRN3gamma) was found to associate with estrogen receptor-alpha (ERalpha) in a ligand-dependent manner. In two-hybrid and pull-down analyses, estradiol induced an interaction between rSTRN3gamma and ERalpha. STRN3gamma protein was found in nuclear extracts from rat uterus and human cell lines. Overexpression of rSTRN3gamma induced a decrease in ERalpha transcriptional activity but had no effect on ERbeta activity. Immunoprecipitation analyses showed that rSTRN3gamma interacts with both the ERalpha and the catalytic subunit of protein phosphatase 2A (PP2A(C)). The transrepressor action of rSTRN3gamma was overcome by okadaic acid, an inhibitor of PP2A(C), and by cotransfection of PP2A(C) siRNA. rSTRN3gamma caused dephosphorylation of ERalpha at serine 118 and this was abrogated by okadaic acid. ERalpha lacking phosphorylation sites at either serine 118 or 167 was insensitive to the corepressor action of rSTRN3gamma. These observations suggest that an rSTRN3gamma-PP2A(C) complex is recruited to agonist-activated ERalpha, thereby leading to its dephosphorylation and inhibiting transcription.

Functional p85alpha gene is required for normal murine fetal erythropoiesis.

In vitro studies suggest that activation of class IA phosphatidylinositol 3 (PI-3) kinase is necessary for normal erythroid cell development. However, when class IA PI-3 kinase-deficient mice were generated by a targeted deletion of the p85alpha regulatory subunit, fetal erythropoiesis was reportedly unaffected. Given the discrepancies between these studies, we performed a more detailed in vivo analysis of class IA PI-3 kinase-deficient embryos. Day-14.5 p85alpha-/- embryos are pale with a marked reduction of mature erythrocytes in their peripheral blood. Further, the absolute number and frequency of both early (erythroid burst-forming unit [BFU-E]) and late erythroid progenitors (erythroid colony-forming unit [CFU-E]) are reduced in p85alpha-/- fetal livers compared with wild-type controls, which is associated with reduced proliferation. Taken together, these data establish an important role for p85alpha and class IA PI-3 kinase in regulating the development of both early and late erythroid progenitors in fetal liver.