Egress of mature murine regulatory T cells from the thymus requires RelA.

The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. In this study, we examined the fate of thymic Tregs in TNF-α/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1-null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1, which is reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remained functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7Rα, which is a possible marker of late-stage mature Tregs. Finally, TA-KO fetal liver chimeric mice developed a neuropilin-1(+) splenic Treg population from TA-KO cells, suggesting that Treg arrest was caused by a lack of RelA in the thymic environment. Taken together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment.

Elevation and characteristics of Rab30 and S100a8/S100a9 expression in an early phase of liver regeneration in the mouse.

Recent studies have revealed that cytokines, including TNFα and IL-6 play key roles in the priming phase of liver regeneration. However, further knowledge of molecular events in the priming phase is needed for more comprehensively understanding the initiation of liver regeneration. In the present study, we attempted to identify additional genes involved in an early phase (2-6 h post partial hepatectomy, PH). The expression of 71 genes was shown to be up-regulated more than 3-fold in the liver at 2 h and 6 h post PH, as compared to 0 h (normal livers) using microarray analysis. Among them, Rab30 and S100a8/S100a9, were identified as novel genes up-regulated over 20-fold at 2 h post PH as compared to normal liver, and were further examined by RT-qPCR to confirm microarray results. Rab30 showed no significant up-regulation in organs other than the liver, whereas S100a8/S100a9 showed significant up-regulation in other organs, such as the lung and spleen at 6 h post PH as compared to those of sham-operated mice, indicating the existence of a different up-regulation machinery between Rab30 and S100a8/S100a9. Their expression was further investigated in the liver at various developmental stages. Rab30 was shown to be expressed only in newborn liver, whereas S100a8/S100a9 was highly expressed in embryo stages, and exhibited the highest levels in newborn liver. These findings imply that Rab30 and S100a8/S100a9 are possibly involved in the functional switch from hematopoiesis support to metabolism in the newborn stage, but might play different roles in liver development. In conclusion, Rab30 and S100a8/S100a9 were indicated to play roles in the initiation of liver regeneration as well as possibly in the functional switch of the liver in the newborn stage.

Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes.

BACKGROUND: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. RESULTS: First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. CONCLUSION: Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.

Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation.

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

Recruitment of katanin p60 by phosphorylated NDEL1, an LIS1 interacting protein, is essential for mitotic cell division and neuronal migration.

LIS1 is mutated in the human neuronal migration defect lissencephaly and along with NDEL1 (formerly NUDEL) participates in the regulation of cytoplasmic dynein function during neuronal development. Targeted disruption of Ndel1 suggested that NDEL1 could have other molecular targets that regulate microtubule organization for proper neuronal migration. To further understanding the molecular mechanism of LIS1 and lissencephaly, we identified the katanin p60 microtubule-severing protein as an additional molecular target of NDEL1. We demonstrate that phosphorylation of NDEL1 by Cdk5 facilitates interaction between NDEL1 and p60, suggesting that P-NDEL1 regulates the distribution of katanin p60. Abnormal accumulation of p60 in nucleus of Ndel1 null mutants supports an essential role of NDEL1 in p60 regulation. Complete loss of NDEL1 or expression of dominant negative mutants of p60 in migrating neurons results in defective migration and elongation of nuclear-centrosomal distance. Our results suggest that NDEL1 is essential for mitotic cell division and neuronal migration not only via regulation of cytoplasmic dynein function but also by modulation of katanin p60 localization and function.

Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression.

In order to verify whether tenascin-C (TN-C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375-GFP), implanted subcutaneously into BALB/cA nude (WT) and TN-C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375-GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell-cell interactions. Both the content of TN-C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375-GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN-C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN-C expression of A375-GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN-C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN-C. These results clearly indicated that the expressions of both TN-C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN-C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.

Tenascin-C expression and splice variant in habu snake venom-induced glomerulonephritis.

Extracellular matrix glycoprotein tenascin-C (TNC) expression is up-regulated in tissue remodeling processes such as tumorigenesis and wound healing. Mouse tenascin-C contains six alternatively spliced domains (A1, A2, A4, B, C, and D) between the fifth and the sixth type III fibronectin domains, which generate large numbers of TNC isoforms. To study TNC isoform variability of wound healing in mice, we induced glomerulonephritis by using Habu snake venom (HSV) and examined alternatively spliced regions by the reverse transcription polymerase chain reaction (RT-PCR) technique. RT-PCR products were separated into seven bands in both healthy and diseased kidneys. Among the seven bands, those containing one or five alternatively spliced domains were mainly up-regulated from 2 days to 1 week after HSV injection. Southern blotting revealed that only domain-D detected all six bands in both healthy and diseased kidneys. Furthermore, only the domain-C transcriptional level did not show an obvious change in progress following HSV injection. These results suggested that (a) the isoforms containing one or five alternatively spliced domains play important roles in the healing process of glomerulonephritis, (b) domain-D is particularly significant in kidney, and (c) domain-C may not play an important role in the healing process of HSV-induced glomerulonephritis.