Compensatory role of neuregulin-1 in diabetic cardiomyopathy.

Diabetes mellitus is a prevalent risk factor for congestive heart failure. Diabetic cardiomyopathy patients present with left ventricular (LV) diastolic dysfunction at an early stage, then systolic dysfunction as the disease progresses. The mechanism underlying the development of diabetic cardiomyopathy has not yet been fully understood. This study aimed to elucidate the mechanisms by which diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy. We hypothesized that the downregulation of cardioprotective factors is involved in the pathogenesis of diabetic cardiomyopathy. LV diastolic dysfunction, but not systolic dysfunction, was observed in type-1 diabetes mellitus model mice 4 weeks after STZ administration (STZ-4W), mimicking the early stage of diabetic cardiomyopathy. Counter to expectations, neuregulin-1 (NRG1) was markedly upregulated in the vascular endothelial cell in the ventricles of STZ-4W mice. To clarify the functional significance of the upregulated NRG1, we blocked its receptor ErbB2 with trastuzumab (TRZ). In STZ-4W mice, TRZ significantly reduced the systolic function without affecting diastolic function and caused a more prominent reduction in Akt phosphorylation levels. These results indicate that the compensatory upregulated NRG1 contributes to maintaining the LV systolic function, which explains why diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy.

Pathogenic Mechanism of Dry Eye-Induced Chronic Ocular Pain and a Mechanism-Based Therapeutic Approach.

Purpose: Dry eye-induced chronic ocular pain is also called ocular neuropathic pain. However, details of the pathogenic mechanism remain unknown. The purpose of this study was to elucidate the pathogenic mechanism of dry eye-induced chronic pain in the anterior eye area and develop a pathophysiology-based therapeutic strategy. Methods: We used a rat dry eye model with lacrimal gland excision (LGE) to elucidate the pathogenic mechanism of ocular neuropathic pain. Corneal epithelial damage, hypersensitivity, and hyperalgesia were evaluated on the LGE side and compared with the sham surgery side. We analyzed neuronal activity, microglial and astrocytic activity, α2δ-1 subunit expression, and inhibitory interneurons in the trigeminal nucleus. We also evaluated the therapeutic effects of ophthalmic treatment and chronic pregabalin administration on dry eye-induced ocular neuropathic pain. Results: Dry eye caused hypersensitivity and hyperalgesia on the LGE side. In the trigeminal nucleus of the LGE side, neuronal hyperactivation, transient activation of microglia, persistent activation of astrocytes, α2δ-1 subunit upregulation, and reduced numbers of inhibitory interneurons were observed. Ophthalmic treatment alone did not improve hyperalgesia. In contrast, continuous treatment with pregabalin effectively ameliorated hypersensitivity and hyperalgesia and normalized neural activity, α2δ-1 subunit upregulation, and astrocyte activation. Conclusions: These results suggest that dry eye-induced hypersensitivity and hyperalgesia are caused by central sensitization in the trigeminal nucleus with upregulation of the α2δ-1 subunit. Here, we showed that pregabalin is effective for treating dry eye-induced ocular neuropathic pain even after chronic pain has been established.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Human SLC15A4 is crucial for TLR-mediated type I interferon production and mitochondrial integrity.

Solute carrier family 15 member 4 (SLC15A4) is an endolysosome-resident amino acid transporter that regulates innate immune responses, and is genetically associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and colitis. SLC15A4-deficient mice showed the amelioration of symptoms of these model diseases, and thus SLC15A4 is a promising therapeutic target of SLE and colitis. For developing a SLC15A4-based therapeutic strategy, understanding human SLC15A4's properties is essential. Here, we characterized human SLC15A4 and demonstrated that human SLC15A4 possessed pH- and temperature-dependent activity for the transportation of dipeptides or tripeptides. Human SLC15A4 localized in LAMP1+ compartments and constitutively associated with Raptor and LAMTORs. We also investigated SLC15A4's role in inflammatory responses using the human plasmacytoid dendritic cell line, CAL-1. Knock down (KD) of the SLC15A4 gene in CAL-1 (SLC15A4-KD CAL-1) impaired Toll-like receptor (TLR) 7/8 or TLR9-triggered type I interferon (IFN-I) production and mTORC1 activity, indicating that human SLC15A4 is critical for TLR7/8/9-mediated inflammatory signaling. We also examined SLC15A4's role in the autophagy response since SLC15A4 loss caused the decrease of mTORC1 activity, which greatly influences autophagy. We found that SLC15A4 was not required for autophagy induction, but was critical for autophagy sustainability. Notably, SLC15A4-KD CAL-1 severely decreased mitochondrial membrane potential in starvation conditions. Our findings revealed that SLC15A4 plays a key role in mitochondrial integrity in human cells, which might benefit immune cells in fulfilling their functions in an inflammatory milieu.

Lysosome biogenesis regulated by the amino-acid transporter SLC15A4 is critical for functional integrity of mast cells.

Mast cells possess specialized lysosomes, so-called secretory granules, which play a key role not only in allergic responses but also in various immune disorders. The molecular mechanisms that control secretory-granule formation are not fully understood. Solute carrier family member 15A4 (SLC15A4) is a lysosome-resident amino-acid/oligopeptide transporter that is preferentially expressed in hematopoietic lineage cells. Here, we demonstrated that SLC15A4 is required for mast-cell secretory-granule homeostasis, and limits mast-cell functions and inflammatory responses by controlling the mTORC1-TFEB signaling axis. In mouse Slc15a4-/- mast cells, diminished mTORC1 activity increased the expression and nuclear translocation of TFEB, a transcription factor, which caused secretory granules to degranulate more potently. This alteration of TFEB function in mast cells strongly affected the FcεRI-mediated responses and IL-33-triggered inflammatory responses both in vitro and in vivo. Our results reveal a close relationship between SLC15A4 and secretory-granule biogenesis that is critical for the functional integrity of mast cells.

Regulation of signal transduction by spatial parameters: a case in NF-κB oscillation.

NF-κB is a transcription factor regulating expression of more than 500 genes, and its dysfunction leads to the autoimmune and inflammatory diseases. In malignant cancer cells, NF-κB is constitutively activated. Thus the elucidation of mechanisms for NF-κB regulation is important for the establishment of therapeutic treatment caused by incorrect NF-κB responses. Cytoplasmic NF-κB translocates to the nucleus by the application of extracellular stimuli such as cytokines. Nuclear NF-κB is known to oscillate with the cycle of 1.5-4.5 h, and it is thought that the oscillation pattern regulates the expression profiles of genes. In this review, first we briefly describe regulation mechanisms of NF-κB. Next, published computational simulations on the oscillation of NF-κB are summarised. There are at least 60 reports on the computational simulation and analysis of NF-κB oscillation. Third, the importance of a 'space' for the regulation of oscillation pattern of NF-κB is discussed, showing altered oscillation pattern by the change in spatial parameters such as diffusion coefficient, nuclear to cytoplasmic volume ratio (N/C ratio), and transport through nuclear membrane. Finally, simulations in a true intracellular space (TiCS), which is an intracellular 3D space reconstructed in a computer with organelles such as nucleus and mitochondria are discussed.

Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly.

Stress granules (SGs) are non-membranous cytoplasmic aggregates of mRNAs and related proteins, assembled in response to environmental stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, chemicals (e.g. arsenite), and viral infections. SGs are hypothesized as a loci of mRNA triage and/or maintenance of proper translation capacity ratio to the pool of mRNAs. In brain ischemia, hippocampal CA3 neurons, which are resilient to ischemia, assemble SGs. In contrast, CA1 neurons, which are vulnerable to ischemia, do not assemble SGs. These results suggest a critical role SG plays in regards to cell fate decisions. Thus SG assembly along with its dynamics should determine the cell fate. However, the process that exactly determines the SG assembly dynamics is largely unknown. In this paper, analyses of experimental data and computer simulations were used to approach this problem. SGs were assembled as a result of applying arsenite to HeLa cells. The number of SGs increased after a short latent period, reached a maximum, then decreased during the application of arsenite. At the same time, the size of SGs grew larger and became localized at the perinuclear region. A minimal mathematical model was constructed, and stochastic simulations were run to test the modeling. Since SGs are discrete entities as there are only several tens of them in a cell, commonly used deterministic simulations could not be employed. The stochastic simulations replicated observed dynamics of SG assembly. In addition, these stochastic simulations predicted a gamma distribution relative to the size of SGs. This same distribution was also found in our experimental data suggesting the existence of multiple fusion steps in the SG assembly. Furthermore, we found that the initial steps in the SG assembly process and microtubules were critical to the dynamics. Thus our experiments and stochastic simulations presented a possible mechanism regulating SG assembly.

Regulation of NF-κB Oscillation by Nuclear Transport: Mechanisms Determining the Persistency and Frequency of Oscillation.

The activated transcription factor NF-κB shuttles between the cytoplasm and the nucleus resulting in the oscillation of nuclear NF-κB (NF-κBn). The oscillation pattern of NF-κBn is implicated in the regulation of gene expression profiles. Using computational models, we previously reported that spatial parameters, such as the diffusion coefficient, nuclear to cytoplasmic volume ratio, transport through the nuclear envelope, and the loci of translation of IκB protein, modified the oscillation pattern of NF-κBn. In a subsequent report, we elucidated the importance of the "reset" of NF-κBn (returning of NF-κB to the original level) and of a "reservoir" of IκB in the cytoplasm. When the diffusion coefficient of IκB was large, IκB stored at a distant location from the nucleus diffused back to the nucleus and "reset" NF-κBn. Herein, we report mechanisms that regulate the persistency and frequency of NF-κBn oscillation by nuclear transport. Among the four parameters of nuclear transport tested in our spatio-temporal computational model, the export of IκB mRNA from the nucleus regulated the persistency of oscillation. The import of IκB to the nucleus regulated the frequency of oscillation. The remaining two parameters, import and export of NF-κB to and from the nucleus, had virtually no effect on the persistency or frequency. Our analyses revealed that lesser export of IκB mRNA allowed NF-κBn to transcript greater amounts of IκB mRNA, which was retained in the nucleus, and was subsequently exported to the cytoplasm, where large amounts of IκB were synthesized to "reset" NF-κBn and drove the persistent oscillation. On the other hand, import of greater amounts of IκB led to an increase in the influx and the efflux of NF-κB to and from the nucleus, resulting in an increase in the oscillation frequency. Our study revealed the importance of nuclear transport in regulating the oscillation pattern of NF-κBn.

Limitation of immune tolerance-inducing thymic epithelial cell development by Spi-B-mediated negative feedback regulation.

Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.

Regulation of nuclear NF-κB oscillation by a diffusion coefficient and its biological implications.

The transcription factor NF-κB shuttles between the cytoplasm and the nucleus, and nuclear NF-κB is known to oscillate with a cycle of 1.5-2.5 h following the application of external stimuli. Oscillation pattern of NF-κB is implicated in regulation of the gene expression profile. In a previous report, we found that the oscillation pattern of nuclear NF-κB in a computational 3D spherical cell was regulated by spatial parameters such as nuclear to cytoplasmic volume ratio, nuclear transport, locus of protein synthesis, and diffusion coefficient. Here we report analyses and a biological implication for the regulation of oscillation pattern by diffusion coefficient. Our analyses show that the "reset" of nuclear NF-κB, defined as the return of nuclear NF-κB to the initial level or lower, was crucial for the oscillation; this was confirmed by the flux analysis. In addition, we found that the distant cytoplasmic location from the nucleus acted as a "reservoir" for storing newly synthesized IκBα. When the diffusion coefficient of proteins was large (≥ 10-11 m2/s), a larger amount of IκBα was stored in the "reservoir" with a large flux by diffusion. Subsequently, stored IκBα diffused back to the nucleus, where nuclear NF-κB was "reset" to the initial state. This initiated the next oscillation cycle. When the diffusion coefficient was small (≤ 10-13 m2/s), oscillation of nuclear NF-κB was not observed because a smaller amount of IκBα was stored in the "reservoir" and there was incomplete "reset" of nuclear NF-κB. If the diffusion coefficient for IκBα was increased to 10-11 m2/s keeping other proteins at 10-13 m2/s, the oscillation was rescued confirming the "reset" and "reservoir" hypothesis. Finally, we showed altered effective value of diffusion coefficient by diffusion obstacles. Thus, organelle crowding seen in stressed cells possibly changes the oscillation pattern by controlling the effective diffusion coefficient.

Identification of transcription factors activated in thymic epithelial cells during embryonic thymus development.

Differentiation of many immune-related cells is controlled by the expression levels and the activation status of transcription factors (TFs). We here describe a method to identify candidate TFs activated during the development of thymic epithelial cells (TECs) in the embryo. RNAs are isolated from fetal thymic organ cultures of wild-type and mutant mice and are subsequently analyzed by using a combination of comprehensive expression analysis and in silico data analysis in order to predict the TFs that might be activated.

Roles of spatial parameters on the oscillation of nuclear NF-κB: computer simulations of a 3D spherical cell.

Transcription factor NF-κB resides in the cytoplasm and translocates to the nucleus by application of extracellular stimuli. It is known that the nuclear NF-κB oscillates and different oscillation patterns lead to different gene expression. Nearly forty reports on modeling and simulation of nuclear NF-κB have been published to date. The computational models reported so far are temporal or two-dimensional, and the discussions on spatial parameters have not been involved or limited. Since spatial parameters in cancer cells such as nuclear to cytoplasmic volume (N/C) ratio are different from normal cells, it is important to understand the relationship between oscillation patterns and spatial parameters. Here we report simulations of a 3D computational model for the oscillation of nuclear NF-κB using A-Cell software. First, we found that the default biochemical kinetic constants used in the temporal model cannot replicate the experimentally observed oscillation in the 3D model. Thus, the default parameters should be changed in the 3D model. Second, spatial parameters such as N/C ratio, nuclear transport, diffusion coefficients, and the location of IκB synthesis were found to alter the oscillation pattern. Third, among them, larger N/C ratios resulted in persistent oscillation of nuclear NF-κB, and larger nuclear transport resulted in faster oscillation frequency. Our simulation results suggest that the changes in spatial parameters seen in cancer cells is one possible mechanism for alteration in the oscillation pattern of nuclear NF-κB and lead to the altered gene expression in these cells.

RANK signaling induces interferon-stimulated genes in the fetal thymic stroma.

Medullary thymic epithelial cells (mTECs) are essential for thymic negative selection to prevent autoimmunity. Previous studies show that mTEC development is dependent on the signal transducers TRAF6 and NIK. However, the downstream target genes of signals controlled by these molecules remain unknown. We performed a microarray analysis on mRNAs down-regulated by deficiencies in TRAF6 or functional NIK in an in vitro organ culture of fetal thymic stromata (2DG-FTOC). An in silico analysis of transcription factor binding sites in plausible promoter regions of differentially expressed genes suggests that STAT1 is involved in TRAF6- and NIK-dependent gene expression. Indeed, the signal of RANK, a TNF receptor family member that activates TRAF6 and NIK, induces the activation of STAT1 in 2DG-FTOC. Moreover, RANK signaling induces the up-regulation of interferon (IFN)-stimulated gene (ISG) expression, suggesting that the RANKL-dependent activation of STAT1 up-regulates ISG expression. The RANKL-dependent expression levels of ISGs were reduced but not completely abolished in interferon α receptor 1-deficient (Ifnar1(-/-)) 2DG-FTOC. Our data suggest that RANK signaling induces ISG expression in both type I interferon-independent and interferon-dependent mechanisms.

TRAF6 directs commitment to regulatory T cells in thymocytes.

Regulatory T cells (Tregs), a subset of CD4(+) helper T cells, are crucial for immunological self-tolerance. Defect in development or function of Tregs results in autoimmune disease in human and mice. Whereas it is known that Tregs mainly develop in the thymus, the molecular mechanism underlying development of Treg is not fully understood. TRAF6-deficient mice showed a severe defect in the Treg development in thymus. In vitro fetal thymic organ culture experiments indicated that the defect is ascribed to the absence of TRAF6 in thymic cells. Moreover, mixed fetal liver transfer experiments revealed that the development of Foxp3(+) cells differentiated from Traf6(-/-) hematopoietic cells was specifically impaired in the thymus, indicating cell-intrinsic requirement for TRAF6 in the Treg development. On the other hand, TRAF6 is not required for the development of conventional CD4(+) T cell. In addition, TGFß-dependent induction of Foxp3 in CD4(+) T cells in vitro was not impaired by the absence of TRAF6. Overall, our data indicate that TRAF6 plays an essential role on the commitment of immature thymocytes to thymic Tregs in cell-intrinsic fashion.

The tumor necrosis factor family receptors RANK and CD40 cooperatively establish the thymic medullary microenvironment and self-tolerance.

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

Developmental stage-dependent collaboration between the TNF receptor-associated factor 6 and lymphotoxin pathways for B cell follicle organization in secondary lymphoid organs.

Signal transduction pathways regulating NF-kappaB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-kappaB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) beta receptor (LtbetaR) signal induction of nonclassical NF-kappaB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtbetaR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

Lack of an inducible effect of dietary soy isoflavones on the mRNA abundance of hepatic cytochrome P-450 isozymes in rats.

Modulation of the activity and content of cytochrome P-450 (CYP) in hepatic microsomes may be important to human health since these enzymes activate and inactivate a wide range of xenobiotics and food components. Regulation of the inducibility of most CYPs involves transcriptional regulation and post-transcriptional mRNA stabilization. We examined in the present study the effect of dietary soy isoflavone (0-300 mg of isoflavone/kg of diet) on the mRNA abundance of rat hepatic CYP1A1, CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A1, CYP3A2 and CYP4A1 by quantitative competitive RT-PCR and real-time monitored RT-PCR. A fermented soy extract containing 155 mg/g of genistein, 127 mg/g of daidzein, and other minor isoflavones was used as the isoflavone source. The dietary soy isoflavone had no affect on the hepatic mRNA abundance of these CYPs. The results by both methods were well matched and indicate that the dietary soy isoflavone did not cause the induction of CYPs by transcriptional step-up regulation or post-transcriptional mRNA stabilization.