Period2 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Perilipin+ embryonic preadipocytes actively proliferate along growing vasculatures for adipose expansion.

Despite the growing interest in adipose tissue as a therapeutic target of metabolic diseases, the identity of adipocyte precursor cells (preadipocytes) and the formation of adipose tissue during embryonic development are still poorly understood. Here, we clarified the identity and dynamic processes of preadipocytes in mouse white adipose tissue during embryogenesis through direct examination, lineage tracing and culture systems. Surprisingly, we found that lipid-lacking but perilipin(+) or adiponectin(+) proliferating preadipocytes started to emerge at embryonic day 16.5, and these cells underwent active proliferation until birth. Moreover, these preadipocytes resided as clusters and were distributed along growing adipose vasculatures. Importantly, the embryonic preadipocytes exhibited considerable coexpression of stem cell markers, such as CD24, CD29 and PDGFRα, and a small portion of preadipocytes were derived from PDGFRß(+) mural cells, in contrast to the adult preadipocytes present in the stromal vascular fraction. Further analyses with in vitro and ex vivo culture systems revealed a stepwise but dynamic regulation of preadipocyte formation and differentiation during prenatal adipogenesis. To conclude, we unraveled the identity and characteristics of embryonic preadipocytes, which are crucial for the formation and expansion of adipose tissue during embryogenesis.

Conditional ablation of LYVE-1+ cells unveils defensive roles of lymphatic vessels in intestine and lymph nodes.

To unveil the organotypic role and vulnerability of lymphatic vessels, we generated a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-Cre/iDTR double-transgenic mouse and ablated LYVE-1-expressing lymphatic vessels in adult mice in a diphtheria toxin (DT)-inducible manner based on selective expression of LYVE-1 in most lymphatic vessels. Strikingly, lymphatic vessels in the small intestine and lymph nodes were rapidly ablated, but lymphatic vessels in the other organs were relatively intact at 24 hours after DT administration. Unexpectedly, LYVE-1-Cre/iDTR mice died of sepsis without visible edema at 24 and 60 hours after DT administration. The cause of death appeared to be related to acute failure of immune surveillance systems in the small intestine and draining lymph nodes. Of note, acute loss of lymphatic lacteals in intestinal villi appeared to trigger distortion of blood capillaries and the whole architecture of the villi, whereas acute loss of lymphatic vessels in lymph nodes caused dysfunction of lymph drainage and abnormal distribution of dendritic cells and macrophages. Thus, intact lymphatic vessels are required for structural and functional maintenance of surrounding tissues in an organotypic manner, at least in the intestine and lymph nodes.

Antibody-secreting cells with a phenotype of Ki-67low, CD138high, CD31high, and CD38high secrete nonspecific IgM during primary hepatitis A virus infection.

Although studies investigating the nature of Ab-secreting cells (ASCs) during acute infection with influenza or dengue virus found that the ASC response was dominated by virus-specific IgG secretion, the Ag specificity and phenotype of ASCs during primary acute viral infection were not identified. To this end, we investigated the nature of ASCs in direct ex vivo assays from patients with acute hepatitis A caused by primary infection with hepatitis A virus (HAV). We found that the frequency of CD27(high)CD38(high) ASCs was markedly increased in the peripheral blood during the acute phase of HAV infection. Moreover, substantial numbers of ASCs were non-HAV-specific and dominantly secreted IgM. We detected HAV-specific ASCs by staining with fluorochrome-tagged HAV-VP1 protein. As compared with HAV-specific ASCs, non-HAV-specific ASCs were Ki-67(low)CD138(high)CD31(high)CD38(high), demonstrating that non-HAV-specific ASCs had a bone marrow plasma cell-like phenotype whereas HAV-specific ASCs had a phenotype typical of circulating plasmablasts. These data suggest that non-HAV-specific ASCs might be mobilized plasma cells from the bone marrow or the spleen, whereas HAV-specific ASCs were newly generated plasmablasts. In this study, we provide evidence that pre-existing plasma cells are released into the circulation and contribute to Ag-nonspecific secretion of IgM during primary HAV infection.

CCCP induces autophagy in an AMPK-independent manner.

AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.

The essential role of FKBP38 in regulating phosphatase of regenerating liver 3 (PRL-3) protein stability.

The phosphatase of regenerating liver-3 (PRL-3) is a member of protein tyrosine phosphatases and whose deregulation is implicated in tumorigenesis and metastasis of many cancers. However, the underlying mechanism by which PRL-3 is regulated is not known. In this study, we identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as an interacting protein of PRL-3 using a yeast two-hybrid system. FKBP38 specifically binds to PRL-3 in vivo, and that the N-terminal region of FKBP38 is crucial for binding with PRL-3. FKBP38 overexpression reduces endogenous PRL-3 expression levels, whereas the depletion of FKBP38 by siRNA increases the level of PRL-3 protein. Moreover, FKBP38 promotes degradation of endogenous PRL-3 protein via protein-proteasome pathway. Furthermore, FKBP38 suppresses PRL-3-mediated p53 activity and cell proliferation. These results demonstrate that FKBP38 is a novel regulator of the oncogenic protein PRL-3 abundance and that alteration in the stability of PRL-3 can have a dramatic impact on cell proliferation. Thus, FKBP38 may play a critical role in tumorigenesis.

Angiogenic role of LYVE-1-positive macrophages in adipose tissue.

Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1(+) macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1(+) macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue.

Immunoproteasome induction is suppressed in hepatitis C virus-infected cells in a protein kinase R-dependent manner.

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8+ cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells. This finding indicated that immunoproteasome induction was impaired at the translational or posttranslational level by HCV infection. Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells.

Transcriptional regulation and life-span modulation of cytosolic aconitase and ferritin genes in C.elegans.

Ferritin is the major iron storage protein regulating cytosolic concentration of iron by storing excess iron. Vertebrate ferritins are heteropolymeric proteins composed of heavy chain and light chain subunits. We have characterized two Caenorhabditis elegans genes (ftn-1 and ftn-2), which encode ferritin homologs showing high degree of similarity to mammalian ferritin heavy chains. Even though these two ferritins are more than 78% identical in amino acid sequence, our data show that expression patterns and responses to iron are quite different. Cytosolic aconitase (aco-1), iron regulatory protein, is known to regulate cellular iron concentration by modulating translation of the ferritin mRNA in addition to its enzymatic activity that converts citrate into iso-citrate. We have shown that the expression levels of aco-1 and ftn-1 genes are both regulated by iron treatment but in opposite ways. Interestingly, mutant animals lacking ACO-1 and FTN-1 show significantly reduced life-span upon iron stress, while N2 and ftn-2 animals show no difference. Our results suggest that ftn-1 and aco-1 are transcriptionally regulated by iron and are important for iron homeostasis affecting life-span upon iron stress conditions in C.elegans.

Crystal structure of Drosophila angiotensin I-converting enzyme bound to captopril and lisinopril.

Angiotensin I-converting enzymes (ACEs) are zinc metallopeptidases that cleave carboxy-terminal dipeptides from short peptide hormones. We have determined the crystal structures of AnCE, a Drosophila homolog of ACE, with and without bound inhibitors to 2.4 A resolution. AnCE contains a large internal channel encompassing the entire protein molecule. This substrate-binding channel is composed of two chambers, reminiscent of a peanut shell. The inhibitor and zinc-binding sites are located in the narrow bottleneck connecting the two chambers. The substrate and inhibitor specificity of AnCE appears to be determined by extensive hydrogen-bonding networks and ionic interactions in the active site channel. The catalytically important zinc ion is coordinated by the conserved Glu395 and histidine residues from a HExxH motif.

Integrative analysis of multiple gene expression profiles applied to liver cancer study.

A statistical method for combining multiple microarray studies has been previously developed by the authors. Here, we present the application of the method to our hepatocellular carcinoma (HCC) data and report new findings on gene expression changes accompanying HCC. From the cross-verification result of our studies and that of published studies, we found that single microarray analysis might lead to false findings. To avoid those pitfalls of single-set analyses, we employed our effect size method to integrate multiple datasets. Of 9982 genes analyzed, 477 significant genes were identified with a false discovery rate of 10%. Gene ontology (GO) terms associated with these genes were explored to validate our method in the biological context with respect to HCC. Furthermore, it was demonstrated that the data integration process increases the sensitivity of analysis and allows small but consistent expression changes to be detected. These integration-driven discoveries contained meaningful and interesting genes not reported in previous expression profiling studies, such as growth hormone receptor, erythropoietin receptor, tissue factor pathway inhibitor-2, etc. Our findings support the use of meta-analysis for a variety of microarray data beyond the scope of this specific application.

Angiopoietin-1 suppresses choroidal neovascularization and vascular leakage.

PURPOSE: To investigate the role of angiopoietin-1 (Ang1) in choroidal neovascularization (CNV) and vascular leakage. METHODS: We generated laser-induced CNV in mice and measured the size of CNV and vascular leakage after intravitreal administration of Ang1. The expressions and distributions of endothelial junctional proteins were analyzed using immunohistochemistry and Western blot. Moreover, we compared the sizes of CNV and vascular leakage in Ang1-overexpressing, Ang1-deficient, and their littermate control mice. In addition, following the transplantation of GFP(+) bone marrow cells into these Ang1-genetically modified mice, we evaluated the recruitment of VEGF-A producing macrophages from the bone marrow after CNV induction. RESULTS: Intravitreal administration of Ang1 was as effective as VEGF-Trap in inhibiting CNV formation. Furthermore, Ang1 suppressed vascular leakage by increasing endothelial junctional proteins, which was more effective than VEGF-Trap. Genetic deletion of Ang1 exacerbated, while overexpression of Ang1 suppressed CNV formation and vascular leakage. We attribute these Ang1-induced, anti-angiogenic, and anti-leakage effects to its inhibitory actions against the recruitment and infiltration of VEGF-A-producing macrophages from bone marrow into the inflammatory lesions. CONCLUSIONS: Ang1 supplementation can be established as a therapeutic strategy to suppress the CNV formation and vascular leakage by inhibiting the recruitment of angiogenic macrophages and tightening the endothelial junctions.

TAK1 regulates autophagic cell death by suppressing the phosphorylation of p70 S6 kinase 1.

There is growing interest in identifying regulators of autophagy. The molecular mechanism underlying transforming growth factor-ß activated kinase 1 (TAK1)-induced autophagy is poorly understood. We found that TAK1 inhibits p70 S6 kinase1 (S6K1) phosphorylation by interfering interaction of raptor with S6K1, thus inducing autophagy. The factors that determine whether autophagy is cytoprotective or cytotoxic have not been fully elucidated. In Drosophila, TAK1 overexpression leads to an impaired eye phenotype despite inhibition of apoptosis, indicating that the phenotype was mainly due to autophagy. Also, TAK1 overexpression increases lactate dehydrogenase (LDH) level in mammalian cells. When treated with autophagy inhibitors, the level of TAK1-induced cytotoxicity or cell death was significantly attenuated, indicating that TAK1 induces cytotoxic autophagic cell death. This study provides the first in vitro and in vivo evidence of TAK1-induced autophagy and we believe that our findings significantly contribute to the understanding of the mechanisms underlying the induction of autophagy.

Immunosenescent CD8+ T cells and C-X-C chemokine receptor type 3 chemokines are increased in human hypertension.

The pathogenic role of T cells in hypertension has been documented well in recent animal studies. However, the existence of T-cell-driven inflammation in human hypertension has not been confirmed. Therefore, we undertook immunologic characterization of T cells from patients with hypertension and measured circulating levels of C-X-C chemokine receptor type 3 chemokines, which are well-known tissue-homing chemokines for T cells. We analyzed immunologic markers on T cells from patients with hypertension by multicolor flow cytometry. We then measured circulating levels of the C-X-C chemokine receptor type 3 chemokines, monokine induced by γ interferon (IFN), IFN γ-induced protein 10, and IFN-inducible T-cell α chemoattractant, in patients with hypertension and in age- and sex-matched control subjects by the cytometric bead array method. In addition, we examined histological features of IFN-inducible T-cell α chemoattractant expression from renal biopsy specimens of patients with hypertensive nephrosclerosis and control subjects. The total T-cell population from patients with hypertension showed an increased fraction of immunosenescent, proinflammatory, cytotoxic CD8(+) T cells. Circulating levels of C-X-C chemokine receptor type 3 chemokines were significantly higher in patients with hypertension than in control subjects. Furthermore, immunohistochemical staining revealed increased expression of the T-cell chemokine, IFN-inducible T-cell α chemoattractant, in the proximal and distal tubules of patients with hypertensive nephrosclerosis. Immunosenescent CD8(+) T cells and C-X-C chemokine receptor type 3 chemokines are increased in human hypertension, suggesting a role for T-cell-driven inflammation in hypertension. A more detailed characterization of CD8(+) T cells may offer new opportunities for the prevention and treatment of human hypertension.

Angiopoietin-1 guides directional angiogenesis through integrin αvß5 signaling for recovery of ischemic retinopathy.

Retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are ischemic retinal diseases caused by insufficient vascular network formation and vascular regression in addition to aberrant angiogenesis. We examined the role of angiopoietin-1 (Ang1) in retinal vascular network formation during postnatal development using Ang1 gain- and loss-of-function mouse models, and tested the effects of intraocular administration of Ang1 in an oxygen-induced retinopathy (OIR) mouse model that mimics cardinal features of ROP and PDR. We observed that Ang1 plays a substantial role in the formation of the retinal vascular network during postnatal development and that Ang1 supplementation can rescue vascular retinopathies by simultaneously promoting healthy vascular network formation and inhibiting subsequent abnormal angiogenesis, vascular leakage, and neuronal dysfunction in the retinas of the OIR model. We attribute these Ang1-induced effects to a dual signaling pathway-Tie2 signaling in the vascular region and integrin αvß5 signaling in the astrocytes. The activation of integrin αvß5 signaling promoted fibronectin accumulation and radial distribution along the sprouting endothelial cells, which consequently stimulated guided angiogenesis in the retina. These findings shed light on the role of Ang1 in the recovery of ischemic retinopathies such as ROP, PDR, and retinal vascular occlusive disease.

Altered expression of phosphatase of regenerating liver gene family in non-small cell lung cancer.

Protein tyrosine phophatases (PTPs) are implicated in the tumorigenesis and metastasis of human cancer. The phosphatase of regenerating liver (PRL) gene family, a subgroup of PTPs is also linked to these processes. In many solid cancers, high levels of PRL-3 expression are related with metastasis and poor prognosis. However, the expression patterns of PRL-1 and -2 have not been explored in lung cancer yet. Thus, we investigated the expression levels of PRL-1, -2 and -3 in the tissues of primary lung cancer patients. The protein expression levels of PRL-2, but not PRL-1 and -3 were increased in cancer tissues. However, there was no correlation between mRNA and protein expression levels of the PRLs. Reporter assays showed that PRLs suppressed the activity of the p21 promoter but promoted AP-1 activity. Furthermore, transfection of PRLs showed significantly increased cell proliferation. Therefore, these results suggest that PRL-2 plays an important role in lung cancer and can be a biomarker of lung cancer, substituting for the function of other PRLs.

The BTB/POZ-ZF transcription factor dPLZF is involved in Ras/ERK signaling during Drosophila wing development.

In Drosophila, broad complex, tramtrack, bric à brac (BTB)/poxvirus and zinc finger (POZ) transcription factors are essential regulators of development. We searched the Drosophila genome for BTB/POZ-ZF domains and discovered an unknown Drosophila gene, dPLZF, which encodes an orthologue of human PLZF. We then characterized the biological function of the dPLZF via genetic interaction analysis. Ectopic expression of dPLZF in the wing induced extra vein formation during wing development in Drosophila. Genetic interactions between dPLZF and Ras or extracellular signal-regulated kinase (ERK) significantly enhanced the formation of vein cells. On the other hand, loss-of-function mutations in dPLZF resulted in a dramatic suppression of the extra and ectopic vein formation induced by elevated Ras/ERK signaling. Moreover, dPLZF activity upregulated the expression of rhomboid (rho) and spitz, which perform crucial functions in vein cell formation in the developing wing. These results indicate that dPLZF is a transcription factor controlled by the Ras/ERK signaling pathway, which is a prominent regulator of vein cell formation during wing development in Drosophila.

Downregulation of p53 by phosphatase of regenerating liver 3 is mediated by MDM2 and PIRH2.

AIMS: The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. MAIN METHODS: To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. KEY FINDINGS: We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. SIGNIFICANCE: These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.

Double antiangiogenic protein, DAAP, targeting VEGF-A and angiopoietins in tumor angiogenesis, metastasis, and vascular leakage.

Two vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage.

Structural diversity of the hagfish variable lymphocyte receptors.

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.