Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Differential effects of reproductive interference by an alien congener on native Taraxacum species

Reproductive interference (RI) has been suggested to play a critical role in native plant displacement by alien congeners. However, although co-existence of native and alien congeners may provide an opportunity to refute the RI hypothesis, few studies have examined such a case. Using a native Japanese dandelion, Taraxacum longeappendiculatum, and a co-existing alien congener, Taraxacum officinale, we tested the hypothesis that differences in RI by the alien between native recipient congeners explain whether a native will co-exist with or be displaced by an alien. We conducted a field survey to investigate the effects of alien relative abundance on T. longeappendiculatum seed set, and a hand-pollination experiment to identify the extent of pollen interference by the alien on T. longeappendiculatum. We compared these results with those obtained previously for another Japanese native species, Taraxacum japonicum, which was displaced by the alien. In our field survey, alien relative abundance had little effect on seed set in nearby T. longeappendiculatum, and hand-pollination with mixed pollen grains produced no substantial decrease in seed set of the native species. Model selection supported these tendencies; the effect of RI by the alien differed between the two native species. Other potential factors, such as resource competition or habitat changes, could not explain the co-existence of T. longeappendiculatum with and exclusion of T. japonicum by the alien in the same explanatory framework. Considering the consistent explanatory power, the findings suggest that RI is a critical mechanism that can determine both co-existence with and displacement of native dandelions by an alien congener.     

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

T2BP, a novel TRAF2 binding protein, can activate NF-kappaB and AP-1 without TNF stimulation.

TRAF2 is a key molecule involved in TNF signaling, which is crucial for the regulation of inflammatory processes. We have identified a novel TRAF2 binding protein, designated as T2BP (TRAF2 binding protein), by a mammalian two-hybrid screening approach. T2BP is a relatively small protein of 184 amino acids, which includes a forkhead-associated domain, the phosphopeptide binding motif. The interaction domain search showed that the TRAF domain in TRAF2 is required for the binding to T2BP whereas almost the entire protein in T2BP binds to TRAF2. The interaction was further confirmed by co-immunoprecipitation. Expression profiling for T2BP and TRAF2 revealed an ubiquitous expression in adult mouse tissues. Overexpression of T2BP in HEK293 cells activated NF-kappaB and AP-1 in a dose dependent manner as well as seen in the TNF-treated control cells. Our results suggest that T2BP is involved in the TNF-mediated signaling by its interaction with TRAF2.     

CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties

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Reproducible Alterations of DNA Methylation at a Specific Population of CpG Islands during Blast Formation of Peripheral Blood Lymphocytes

We investigated the changes in the methylation patterns of CpGislands associated with blast formation of human peripheralblood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2),using restriction landmark genomic scanning with a methylation-sensitiverestriction enzyme (RLGS-M) system. Of about 2,100 Not I spot/lociwhich were analyzed, only 10 showed changes, whereas drasticchanges have been observed in cases of malignant and SV40 transformation.These changes were highly reproducible for samples from boththe same and different individuals. Even the timing of the changesafter cultivation was the same. Thus, we concluded that at leastthe genomic DNA methylation state in vivo was essentially retainedin T blast cells activated in vitro by induction with IL-2 andanti-CD3, which are commonly used in biological experimentsas well as clinical diagnosis and therapy.     

High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327–336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA.