We purified human plasma lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K m value for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.Recent rapid advance in research on phospholipidic mediators has revealed new members that have diverse sets of signaling through their specific G-protein-coupled receptors (1-3). Particularly, lysophosphatidic acid (LPA, 1 radyl-sn-glycerol-3-phosphate) has received interest because of its identification as an active component in platelet-aggregating activity and vasoactivity in incubated animal sera and plasma (4). Later, its principle effects were found to be growth-related; that is, cellular proliferation, alterations in differentiation, and suppression of apoptosis (2-5). Other important effects are its cytoskeletal response, cell aggregation, contraction, adhesion, or chemotaxis (1-5). Its diverse activities appear to be regulated by the relative distribution of its specific receptors in a family of endothelial differentiation genes (EDG), desensitization of the receptors, and the local concentration of LPA determined by balance between LPA-producing and -degrading enzyme activities (1-6). Although LPA is produced intracellularly in phospholipid biosynthesis and turnover, this should be differentiated from its actual pool as a first messenger unless there are efficient mechanisms for its release into extracellular fluid or leaflets of plasma membranes (3-6). In this sense, secretory or ecto-type LPA-producing enzymes can supply LPA directly to its receptors. The first study on extracellular production of LPA was our own, detecting lysophospholipase D (lysoPLD) activity that produces bioactive LPA from lysophosphatidylcholine (LPC) in rat plasma (7). Its distinct substrate specificity and cation requirement from known intracellular lysoPLD activity suggested that it is a hitherto uncharacterized metalloenzyme (7-9). To understand its physiological and pathological significance, the identity of lysoPLD activity in blood circulation should be clarified. Therefore we purified and characterized lysoPLD in human plasma and obtained strong evid...

Thymocyte trafficking has an important role in thymic selection. Here we show that the Hippo homologue mst1 is required for thymocyte migration and antigen recognition by LFA-1 and ICAm-1 within the medulla. using two-photon imaging of thymic tissues, we found that highly motile mature thymocytes arrest and are activated in the vicinity of rare populations of Aire + ICAm-1 hi medullary thymic epithelia in a negatively selecting environment. notably, mst1 deficiency or blocking the cell adhesion molecules LFA-1 and ICAm-1 results in inefficient migration and antigen recognition of CD4 + thymocytes within the medulla. Consistent with these defects, thymocyte selection is impaired in Mst1 − / − mice, which display T cell-dependent inflammatory infiltrates in multiple organs and develop autoantibodies. our results suggest that mst1 has a key role in regulating thymocyte self-antigen recognition in the medulla.

Background: 20a-Hydroxysteroid dehydrogenase (HSD) is a member of the aldo-keto reductase (AKR) superfamily and catalyses the reaction of progesterone to the inactive form 20a-hydroxyprogesterone. Progesterone plays an important role in the maintenance of pregnancy, and, in rodents, plasma progesterone levels decrease abruptly just before parturition. The induction of 20a-HSD is thought to be responsible for the decrease in plasma progesterone at term. High homology between human 20a-HSD [AKR 1C1] cDNA with other AKRs had caused dif®culty in gene isolation and expression analysis. Thus, the metabolism of progesterone in the human reproductive system remained unclear.

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1 Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 mM) induced sustained contractions with no increase in [Ca 2 þ ] i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser 19 . 2 The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 mM). LY333531 (1 mM), a specific inhibitor of PKCb, also inhibited the PDBu-induced contraction in the pregnant myometrium. 3 In the pregnant myometrium permeabilized with a-toxin, PDBu increased the contractions induced at fixed Ca 2 þ concentration (0.3 mM) both in nonpregnant and pregnant myometria, indicating Ca 2 þ sensitization of contractile elements. 4 Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (a, b, g), novel PKC (d, e, y), and atypical PKC (z but not i and l). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of b isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. 5 CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. 6 These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the b PKC isoform and CPI-17.

In human endometrium, cytokines and growth factors that vary periodically during the menstrual cycles have been suggested to play various roles in uterine function. In the present study, differential gene expression in human endometrium between the proliferative and the secretory phases was investigated by using a human cDNA expression array system. Human interleukin (IL)-15 was identified as an up-regulated transcription product during the secretory phase, in comparison with the proliferative phase, and therefore its expression in human uterus was examined by Northern blot analysis. In human endometrium, expression of IL-15 mRNA significantly increased during the secretory phase compared with the proliferative phase (P < 0.01). The most abundant expression of IL-15 mRNA during the menstrual cycle was observed in the midsecretory phase. In the first trimester pregnancy, the expression of IL-15 mRNA in the decidua was significantly higher than that in the chorionic villi (P < 0.01). By using an in-vitro decidualization with human endometrial stromal cells, it was demonstrated that the expression of IL-15 mRNA is up-regulated during progesterone-induced decidualization. These results suggest that IL-15 plays a role in uterine function during pregnancy, as well as during the menstrual cycle.

Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1–ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mislocalized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.

Hypoxia simultaneously acts to increase VEGF via HIF-1α and to decrease SDF-1 in a HIF-1α-independent manner in ESCs. These results indicate a potential mechanism for the action of hypoxic conditions that could influence angiogenesis in the human endometrium.