Inhibition of Wnt/beta-Catenin Signaling by p38 MAP Kinase Inhibitors Is Explained by Cross-Reactivity with Casein Kinase I delta/epsilon

SummaryWnt/β-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated β-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/β-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated β-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ɛ, which are known activators of Wnt/β-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of β-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/β-catenin signaling

Discovery of Novel Small Molecule Activators of β-Catenin Signaling

Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling

Circulating lipoprotein (a) and all-cause and cause-specific mortality: a systematic review and dose-response meta-analysis.

AIMS To investigate the association between circulating lipoprotein(a) (Lp(a)) and risk of all-cause and cause-specific mortality in the general population and in patients with chronic diseases, and to elucidate the dose-response relations. METHODS AND RESULTS We searched literature to find prospective studies reporting adjusted risk estimates on the association of Lp(a) and mortality outcomes. Forty-three publications, reporting on 75 studies (957,253 participants), were included. The hazard ratios (HRs) and 95% confidence intervals (95%CI ) for the top versus bottom tertile of Lp(a) levels and risk of all-cause mortality were 1.09 (95%CI: 1.01-1.18, I2: 75.34%, n = 19) in the general population and 1.18 (95%CI: 1.04-1.34, I2: 52.5%, n = 12) in patients with cardiovascular diseases (CVD). The HRs for CVD mortality were 1.33 (95%CI: 1.11-1.58, I2: 82.8%, n = 31) in the general population, 1.25 (95%CI: 1.10-1.43, I2: 54.3%, n = 17) in patients with CVD and 2.53 (95%CI: 1.13-5.64, I2: 66%, n = 4) in patients with diabetes mellitus. Linear dose-response analyses revealed that each 50 mg/dL increase in Lp(a) levels was associated with 31% and 15% greater risk of CVD death in the general population and in patients with CVD. No non-linear dose-response association was observed between Lp(a) levels and risk of all-cause or CVD mortality in the general population or in patients with CVD (Pnonlinearity > 0.05). CONCLUSION This study provides further evidence that higher Lp(a) levels are associated with higher risk of all-cause mortality and CVD-death in the general population and in patients with CVD. These findings support the ESC/EAS Guidelines that recommend Lp(a) should be measured at least once in each adult person's lifetime, since our study suggests those with higher Lp(a) might also have higher risk of mortality

TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

BACKGROUND: TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication. METHODS: To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells. RESULTS: Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G(2)/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G(2)/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells. CONCLUSION: These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis

Evolutionary patterns of two major reproduction candidate genes (Zp2 and Zp3) reveal no contribution to reproductive isolation between bovine species

<p>Abstract</p> <p>Background</p> <p>It has been established that mammalian egg zona pellucida (ZP) glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing the sperm acrosome reaction, and preventing polyspermy. In mammals, ZP apparently represents a barrier to heterospecific fertilization and thus probably contributes to reproductive isolation between species. The evolutionary relationships between some members of the tribe Bovini are complex and highly debatable, particularly, those involving <it>Bos </it>and <it>Bison </it>species for which interspecific hybridization is extensively documented. Because reproductive isolation is known to be a major precursor of species divergence, testing evolutionary patterns of ZP glycoproteins may shed some light into the speciation process of these species. To this end, we have examined intraspecific and interspecific genetic variation of two ZP genes (<it>Zp2 </it>and <it>Zp3</it>) for seven representative species (111 individuals) from the Bovini tribe, including five species from <it>Bos </it>and <it>Bison</it>, and two species each from genera <it>Bubalus </it>and <it>Syncerus</it>.</p> <p>Results</p> <p>A pattern of low levels of intraspecific polymorphism and interspecific divergence was detected for the two sequenced fragments each for <it>Zp2 </it>and <it>Zp3</it>. At intraspecific level, none of neutrality tests detected deviations from neutral equilibrium expectations for the two genes. Several haplotypes in both genes were shared by multiple species from <it>Bos </it>and <it>Bison</it>.</p> <p>Conclusions</p> <p>Here we argue that neither ancestral polymorphism nor introgressive hybridization alone can fully account for haplotype sharing among species from <it>Bos </it>and <it>Bison</it>, and that both scenarios have contributed to such a pattern of haplotype sharing observed here. Additionally, codon-based tests revealed strong evidence for purifying selection in the <it>Zp3 </it>coding haplotype sequences and weak evidence for purifying selection in the <it>Zp2 </it>coding haplotype sequences. Contrary to a general genetic pattern that genes or genomic regions contributing to reproductive isolation between species often evolve rapidly and show little or no gene flow between species, these results demonstrate that, particularly, those sequenced exons of the <it>Zp2 </it>and the <it>Zp3 </it>did not show any contribution to reproductive isolation between the bovine species studied here.</p

Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling Pathway

While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway

ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae

<p>Abstract</p> <p>Background</p> <p>Recent progress in exploring the Y-chromosome gene content in humans, mice and cats have suggested that "autosome-to-Y" transposition of the male fertility genes is a recurrent theme during the mammalian Y-chromosome evolution. These transpositions are lineage-dependent. The purpose of this study is to investigate the lineage-specific Y-chromosome genes in bovid.</p> <p>Results</p> <p>We took a direct testis cDNA selection strategy and discovered two novel gene families, <it>ZNF280BY </it>and <it>ZNF280AY</it>, on the bovine (<it>Bos taurus</it>) Y-chromosome (BTAY), which originated from the transposition of a gene block on the bovine chromosome 17 (BTA17) and subsequently amplified. Approximately 130 active <it>ZNF280BY </it>loci (and ~240 pseudogenes) and ~130 pseudogenized <it>ZNF280AY </it>copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that both gene families fit with the "birth-and-death" model of evolution. The active <it>ZNF280BY </it>loci share high sequence similarity and comprise three major genomic structures, resulted from insertions/deletions (indels). Assembly of a 1.2 Mb BTAY sequence in the MSY ampliconic region demonstrated that <it>ZNF280BY </it>and <it>ZNF280AY</it>, together with <it>HSFY </it>and <it>TSPY </it>families, constitute the major elements within the repeat units. The <it>ZNF280BY </it>gene family was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of <it>ZNF280BY </it>were differentially expressed up to 60-fold. Interestingly, different copies of the <it>ZNF280AY </it>pseudogenes were also found to differentially express up to 10-fold. However, expression level of the <it>ZNF280AY </it>pseudogenes was almost 6-fold lower than that of the <it>ZNF280BY </it>genes. <it>ZNF280BY </it>and <it>ZNF280AY </it>gene families are present in bovid, but absent in other mammalian lineages.</p> <p>Conclusions</p> <p><it>ZNF280BY </it>and <it>ZNF280AY </it>are lineage-specific, multi-copy Y-gene families specific to <it>Bovidae</it>, and are derived from the transposition of an autosomal gene block. The temporal and spatial expression patterns of <it>ZNF280BY</it>s in testis suggest a role in spermatogenesis. This study offers insights into the genomic organization of the bovine MSY and gene regulation in spermatogenesis, and provides a model for studying evolution of multi-copy gene families in mammals.</p

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans