Fumihiko Furuya

Enterovirus Infection, CXC Chemokine Ligand 10 (CXCL10), and CXCR3 Circuit: A Mechanism of Accelerated β-Cell Failure in Fulminant Type 1 Diabetes

OBJECTIVE Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated β-cell failure are unclear. RESEARCH DESIGN AND METHODS Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2–5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR. RESULTS Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor–bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-γ and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including β-cells and α-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells. CONCLUSIONS These results strongly suggest the presence of a circuit for the destruction of β-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-γ and CXCL10 in β-cells. CXCL10 secreted from β-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-γ in the islets, not only damaging β-cells but also accelerating CXCL10 generation in residual β-cells and thus further activating cell-mediated autoimmunity until all β-cells have been destroyed.

Aberrant accumulation of PTTG1 induced by a mutated thyroid hormone β receptor inhibits mitotic progression

Overexpression of pituitary tumor-transforming 1 (PTTG1) is associated with thyroid cancer. We found elevated PTTG1 levels in the thyroid tumors of a mouse model of follicular thyroid carcinoma (TRbeta(PV/PV) mice). Here we examined the molecular mechanisms underlying elevated PTTG1 levels and the contribution of increased PTTG1 to thyroid carcinogenesis. We showed that PTTG1 was physically associated with thyroid hormone beta receptor (TRbeta) as well as its mutant, designated PV. Concomitant with thyroid hormone-induced (T3-induced) degradation of TRbeta, PTTG1 proteins were degraded by the proteasomal machinery, but no such degradation occurred when PTTG1 was associated with PV. The degradation of PTTG1/TRbeta was activated by the direct interaction of the liganded TRbeta with steroid receptor coactivator 3 (SRC-3), which recruits proteasome activator PA28gamma. PV, which does not bind T3, could not interact directly with SRC-3/PA28gamma to activate proteasome degradation, resulting in elevated PTTG1 levels. The accumulated PTTG1 impeded mitotic progression in cells expressing PV. Our results unveil what we believe to be a novel mechanism by which PTTG1, an oncogene, is regulated by the liganded TRbeta. The loss of this regulatory function in PV led to an aberrant accumulation of PTTG1 disrupting mitotic progression that could contribute to thyroid carcinogenesis.

Impaired adipogenesis caused by a mutated thyroid hormone α1 receptor

ABSTRACT Thyroid hormone (T3) is critical for growth, differentiation, and maintenance of metabolic homeostasis. Mice with a knock-in mutation in the thyroid hormone receptor α gene (TRα1PV) were created previously to explore the roles of mutated TRα1 in vivo. TRα1PV is a dominant negative mutant with a frameshift mutation in the carboxyl-terminal 14 amino acids that results in the loss of T3 binding and transcription capacity. Homozygous knock-in TRα1PV/PV mice are embryonic lethal, and heterozygous TRα1PV/+ mice display the striking phenotype of dwarfism. These mutant mice provide a valuable tool for identifying the defects that contribute to dwarfism. Here we show that white adipose tissue (WAT) mass was markedly reduced in TRα1PV/+ mice. The expression of peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipogenesis, was repressed at both mRNA and protein levels in WAT of TRα1PV/+ mice. Moreover, TRα1PV acted to inhibit the transcription activity of PPARγ by competition with PPARγ for binding to PPARγ response elements and for heterodimerization with the retinoid X receptors. The expression of TRα1PV blocked the T3-dependent adipogenesis of 3T3-L1 cells and repressed the expression of PPARγ. Thus, mutations of TRα1 severely affect adipogenesis via cross talk with PPARγ signaling. The present study suggests that defects in adipogenesis could contribute to the phenotypic manifestation of reduced body weight in TRα1PV/+ mice.

Nongenomic activation of phosphatidylinositol 3-kinase signaling by thyroid hormone receptors

Thyroid hormone (T3) is critical in growth, development, differentiation, and maintenance of metabolic homeostasis. Recent studies suggest that thyroid hormone receptors (TRs) not only mediate the biological activities of T3 via nucleus-initiated transcription, but also could act via nongenomic pathways. The striking phenotype of thyroid cancer exhibited by a knockin mutant mouse that harbors a dominant negative TRbeta mutant (TRbeta(PV/PV) mouse) allows the elucidation of novel oncogenic activity of a TRbeta mutant (PV) via extra-nuclear actions. PV physically interacts with the regulatory p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) to activate the downstream AKT-mammalian target of rapamycin (mTOR) and p70(S6K) and PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathways. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. Remarkably, a nuclear receptor corepressor (NCoR) was found to regulate the PV-activated PI3K signaling by competing with PV for binding to the C-terminal SH2 domain of p85alpha. Over-expression of NCoR in thyroid tumor cells of TRbeta(PV/PV) mice reduces AKT-mTOR-p70(S6K) signaling. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells leads to over-activated PI3K-AKT signaling to increase cell proliferation and motility. Furthermore, NCoR protein levels are significantly lower in thyroid tumor cells than in wild type thyrocytes, allowing more effective binding of PV to p85alpha to activate PI3K signaling, thereby contributing to tumor progression. Thus, PV, an apo-TRbeta, could act via direct protein-protein interaction to mediate critical oncogenic actions. These studies also uncovered a novel extra-nuclear role of NCoR in modulating the nongenomic actions of a mutated TRbeta in controlling thyroid carcinogenesis.

RIG-I– and MDA5-Initiated Innate Immunity Linked With Adaptive Immunity Accelerates β-Cell Death in Fulminant Type 1 Diabetes

OBJECTIVE The contribution of innate immunity responsible for aggressive β-cell destruction in human fulminant type 1 diabetes is unclear. RESEARCH DESIGN AND METHODS Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid–inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes. RESULTS RIG-I was strongly expressed in β-cells in all three pancreata infected with enterovirus. Melanoma differentiation–associated gene-5 was hyperexpressed in islet cells, including β- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-β were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid–receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in β-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes. CONCLUSIONS These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive β-cell toxicity in fulminant type 1 diabetes.

Liganded Thyroid Hormone Receptor-α Enhances Proliferation of Pancreatic β-Cells

Failure of the functional pancreatic β-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is important for β-cell proliferation in adult animals. In rat pancreatic β-cell lines (RIN5F), treatment with 100 nm thyroid hormone (triiodothyronine, T3) enhances cell proliferation. This result suggests that T3 is required for β-cell proliferation or replication. To identify the role of thyroid hormone receptor α (TRα) in the processes of β-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTRα. Infection with AdTRα to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTRα-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nm T3. Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTRα-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTRα led to the restoration of islet function and to an increase in the β-cell mass. These results support the hypothesis that liganded TRα plays a critical role in β-cell replication and in expansion of the β-cell mass during postnatal development. Thus, liganded TRα may be a target for therapeutic strategies that can induce the expansion and regeneration of β-cells.

Activation of Phosphatidylinositol 3-Kinase Signaling Promotes Aberrant Pituitary Growth in a Mouse Model of Thyroid-Stimulating Hormone-Secreting Pituitary Tumors

TSH-secreting pituitary tumors (TSHomas) are pituitary tumors that constitutively secrete TSH. Molecular mechanisms underlying this abnormality are largely undefined. We recently created a knock-in mutant mouse harboring a mutation (denoted as PV) in the thyroid hormone receptor-beta gene (TRbeta(PV/PV) mouse). As these mice age, they spontaneously develop TSHomas. Using this mouse model, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the pathogenesis of TSHomas. Concurrent with aberrant growth of pituitaries, AKT and its downstream effectors, mammalian target rapamycin and p70(S6K), were activated to contribute to increased cell proliferation and pituitary growth. In addition, activation of AKT led to decreased apoptosis by inhibiting proapoptotic activity of Bcl-2-associated death promoter, further contributing to the aberrant cell proliferation. These results suggest an activated PI3K-AKT pathway could underscore tumorigenesis, raising the possibility that this pathway could be a potential therapeutic target in TSHomas. Indeed, TRbeta(PV/PV) mice treated with a PI3K-specific inhibitor, LY294002, showed a significant decrease in pituitary growth. The progrowth signaling via AKT-mammalian target rapamycin-p70(S6K) and cyclin D1/cyclin-dependent kinase were inhibited, and proapoptotic activity of Bcl-2-associated death promoter was increased by LY294002 treatment. Thus, activation of the PI3K-AKT pathway mediates, at least in part, the aberrant pituitary growth, and the intervention of this signaling pathway presents a novel therapeutic opportunity for TSHomas.

Absorption of radionuclides from the Fukushima nuclear accident by a novel algal strain.

Large quantities of radionuclides have leaked from the Fukushima Daiichi Nuclear Power Plant into the surrounding environment. Effective prevention of health hazards resulting from radiation exposure will require the development of efficient and economical methods for decontaminating radioactive wastewater and aquatic ecosystems. Here we describe the accumulation of water-soluble radionuclides released by nuclear reactors by a novel strain of alga. The newly discovered green microalgae, Parachlorella sp. binos (Binos) has a thick alginate-containing extracellular matrix and abundant chloroplasts. When this strain was cultured with radioiodine, a light-dependent uptake of radioiodine was observed. In dark conditions, radioiodine uptake was induced by addition of hydrogen superoxide. High-resolution secondary ion mass spectrometry (SIMS) showed a localization of accumulated iodine in the cytosol. This alga also exhibited highly efficient incorporation of the radioactive isotopes strontium and cesium in a light-independent manner. SIMS analysis showed that strontium was distributed in the extracellular matrix of Binos. Finally we also showed the ability of this strain to accumulate radioactive nuclides from water and soil samples collected from a heavily contaminated area in Fukushima. Our results demonstrate that Binos could be applied to the decontamination of iodine, strontium and cesium radioisotopes, which are most commonly encountered after nuclear reactor accidents.

Dual Functions of the Steroid Hormone Receptor Coactivator 3 in Modulating Resistance to Thyroid Hormone

ABSTRACT Mutations of the thyroid hormone receptor β (TRβ) gene cause resistance to thyroid hormone (RTH). RTH is characterized by increased serum thyroid hormone associated with nonsuppressible thyroid-stimulating hormone (TSH) and impaired growth. It is unclear how the actions of TRβ mutants are modulated in vivo to affect the manifestation of RTH. Using a mouse model of RTH that harbors a knockin mutation of the TRβ gene (TRβPV mouse), we investigated the effect of the steroid hormone receptor coactivator 3 (SRC-3) on RTH. In TRβPV mice deficient in SRC-3, dysfunction of the pituitary-thyroid axis and hypercholesterolemia was lessened, but growth impairment of RTH was worsened. The lessened dysfunction of the pituitary-thyroid axis was attributed to a significant decrease in growth of the thyroid and pituitary. Serum insulin-like growth factor 1 (IGF-1) was further reduced in TRβPV mice deficient in SRC-3. This effect led to reduced signaling of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway that is known to mediate cell growth and proliferation. Thus, SRC-3 modulates RTH by at least two mechanisms, one via its role as a receptor coregulator and the other via its growth regulatory role through the IGF-1/PI3K/AKT/mTOR signaling.

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

Background: One goal of diabetic regenerative medicine is to convert mature pancreatic acinar cells into insulin-producing cells. Results: Ligand-bound thyroid hormone receptor α (TRα), which interacts with p85α, induces phosphatidylinositol 3-kinase (PI3K) signaling and insulin expression. Conclusion: PI3K signaling must be activated for TRα-induced reprogramming of pancreatic acinar cells. Significance: TRα is critical for postnatal expansion of the β-cell mass. One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.