[Onset of Testicular Plasmacytoma in a Patient with Refractory Multiple Myeloma].

A 72-year-old male patient, who had been on chemotherapy for the treatment of IgG-λ multiple myeloma, presented an enlargement of the testis 3 years and 5 months after the diagnosis. High orchiectomy was then performed, leading to the diagnosis of plasmacytoma. Due to residual disease, treatment with a combination of isatuximab and dexamethasone was initiated. The patient is currently under follow-up without recurrence. While testicular tumors are difficult to diagnose by imaging studies alone and extramedullary plasmacytomas rarely occur in the testis, pathological assessment is critical for treatment planning.

Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin.

Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined fluorescence of ZnAF-2 DA staining as a marker of intact mossy fibers and increased fluorescence of Fluoro-Jade B staining as a marker of degenerated neurons during the initial 2 to 5 days after the administration with later ameliorations within 30 days in the hippocampal dentate gyrus (DG) and CA3 region in mice. On immunoblotting analysis, both GABABR1 and GABABR2 subunit levels increased during 15 to 30 days after TMT along with significant decreases in glutamatergic GluA1 and GluA2/3 receptor subunit levels during 2 to 7 days in the DG, but not in other hippocampal regions such as CA1 and CA3 regions. Immunohistochemical analysis revealed the constitutive and inducible expression of GABABR2 subunit in cells immunoreactive to an astrocytic marker as well as neuronal markers in the DG with the absence of neither GABABR1a nor GABABR1b subunit from cells positive to an astrocytic marker. These results suggest that both GABABR1 and GABABR2 subunits may be up-regulated in cells other than neurons and astroglia in the DG at a late stage of TMT intoxication in mice.

The MAPK Erk5 is necessary for proper skeletogenesis involving a Smurf-Smad-Sox9 molecular axis.

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.

[Primary Diffuse Large B-Cell Lymphoma of the Testis Developed during the Course of Mycosis Fungoides-A Case Report].

We report the case of an 84-year-old man who developed primary diffuse large B-cell lymphoma of the testes during the course of mycosis fungoides treated with topical medication. He was referred to our hospital due to bilateral testicular masses, and bilateral high orchiectomy was performed. A pathological diagnosis of diffuse large B-cell lymphoma was made after an examination of the surgical specimen. Rituximab-combined miniCHOP chemotherapy with prophylactic intrathecal injection resulted in complete remission without recurrence 1 year after diagnosis. People with mycosis fungoides are known to be at a higher risk of secondary malignancies than healthy individuals; hence, a pathological examination is important to confirm the diagnosis.

Selective Upregulation by Theanine of Slc38a1 Expression in Neural Stem Cell for Brain Wellness.

Theanine is an amino acid abundant in green tea with an amide moiety analogous to glutamine (GLN) rather than glutamic acid (Glu) and GABA, which are both well-known as amino acid neurotransmitters in the brain. Theanine has no polyphenol and flavonoid structures required for an anti-oxidative property as seen with catechins and tannins, which are more enriched in green tea. We have shown marked inhibition by this exogenous amino acid theanine of the uptake of [3H]GLN, but not of [3H]Glu, in rat brain synaptosomes. Beside a ubiquitous role as an endogenous amino acid, GLN has been believed to be a main precursor for the neurotransmitter Glu sequestered in a neurotransmitter pool at glutamatergic neurons in the brain. The GLN transporter solute carrier 38a1 (Slc38a1) plays a crucial role in the incorporation of extracellular GLN for the intracellular conversion to Glu by glutaminase and subsequent sequestration at synaptic vesicles in neurons. However, Slc38a1 is also expressed by undifferentiated neural progenitor cells (NPCs) not featuring a neuronal phenotype. NPCs are derived from a primitive stem cell endowed to proliferate for self-renewal and to commit differentiation to several daughter cell lineages such as neurons, astrocytes, and oligodendrocytes. In vitro culture with theanine leads to the marked promotion of the generation of new neurons together with selective upregulation of Slc38a1 transcript expression in NPCs. In this review, we will refer to a possible novel neurogenic role of theanine for brain wellness through a molecular mechanism relevant to facilitated neurogenesis with a focus on Slc38a1 expressed by undifferentiated NPCs on the basis of our accumulating findings to date.

Genetic analysis of Runx2 function during intramembranous ossification.

Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation and its haploinsufficiency leads to cleidocranial dysplasia because of a defect in osteoblast differentiation during bone formation through intramembranous ossification. The cellular origin and essential period for Runx2 function during osteoblast differentiation in intramembranous ossification remain poorly understood. Paired related homeobox 1 (Prx1) is expressed in craniofacial mesenchyme, and Runx2 deficiency in cells of the Prx1 lineage (in mice referred to here as Runx2prx1 (-/-)) resulted in defective intramembranous ossification. Runx2 was heterogeneously expressed in Prx1-GFP(+) cells located at the intrasutural mesenchyme in the calvaria of transgenic mice expressing GFP under the control of the Prx1 promoter. Double-positive cells for Prx1-GFP and stem cell antigen-1 (Sca1) (Prx1(+)Sca1(+) cells) in the calvaria expressed Runx2 at lower levels and were more homogeneous and primitive than Prx1(+)Sca1(-) cells. Osterix (Osx) is another transcriptional determinant of osteoblast lineages expressed by osteoblast precursors; Osx is highly expressed by Prx1(-)Runx2(+) cells at the osteogenic front and on the surface of mineralized bone in the calvaria. Runx2 deficiency in cells of the Osx lineage (in mice referred to here as Runx2osx (-/-)) resulted in severe defects in intramembranous ossification. These findings indicate that the essential period of Runx2 function in intramembranous ossification begins at the Prx1(+)Sca1(+) mesenchymal stem cell stage and ends at the Osx(+)Prx1(-)Sca1(-) osteoblast precursor stage.

Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction.

Circadian rhythm disturbances are well established in neurological diseases. However, how these disruptions cause homeostatic imbalances remains poorly understood. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) is a major circadian clock transcriptional activator, and Bmal1 deficiency in male Bmal1nestin-/- mice induced marked astroglial activation without affecting the number of astrocytes in the brain and spinal cord. Bmal1 deletion caused blood-brain barrier (BBB) hyperpermeability with an age-dependent loss of pericyte coverage of blood vessels in the brain. Using Nestin-green fluorescent protein (GFP) transgenic mice, we determined that pericytes are Nestin-GFP+ in the adult brain. Bmal1 deletion caused Nestin-GFP+ pericyte dysfunction, including the downregulation of platelet-derived growth factor receptor ß (PDGFRß), a protein necessary for maintaining BBB integrity. Knockdown of Bmal1 downregulated PDGFRß transcription in the brain pericyte cell line. Thus, the circadian clock component Bmal1 maintains BBB integrity via regulating pericytes.SIGNIFICANCE STATEMENT Circadian rhythm disturbances may play a role in neurodegenerative disorders, such as Alzheimer's disease. Our results revealed that one of the circadian clock components maintains the integrity of the blood-brain barrier (BBB) by regulating vascular-embedded pericytes. These cells were recently identified as a vital component for the control of BBB permeability and cerebral blood flow. Our present study demonstrates the involvement of circadian clock component Bmal1 in BBB homeostasis and highlights the role of Bmal1 dysfunction in multiple neurological diseases.

Alleviation by GABAB Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to N-Methyl-D-aspartate.

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.

The intrinsic microglial clock system regulates interleukin-6 expression.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

An L-Glutamine Transporter Isoform for Neurogenesis Facilitated by L-Theanine.

L-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to L-glutamine (L-GLN) rather than L-glutamic acid (L-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. L-theanine markedly inhibits [3H]L-GLN uptake without affecting [3H]L-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to L-theanine, upregulation of the L-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by L-theanine. The green tea amino acid L-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid L-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog L-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of L-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.

ATF3 deficiency in chondrocytes alleviates osteoarthritis development.

Activating transcription factor 3 (Atf3) has been implicated in the pathogenesis of various diseases, including cancer and inflammation, as well as in the regulation of cell proliferation and differentiation. However, the involvement of Atf3 in developmental skeletogenesis and joint disease has not been well studied to date. Here, we show that Atf3 is a critical mediator of osteoarthritis (OA) development through its expression in chondrocytes. ATF3 expression was markedly up-regulated in the OA cartilage of both mice and humans. Conditional deletion of Atf3 in chondrocytes did not result in skeletal abnormalities or affect the chondrogenesis, but alleviated the development of OA generated by surgically inducing knee joint instability in mice. Inflammatory cytokines significantly up-regulated Atf3 expression through the nuclear factor-kB (NF-kB) pathway, while cytokine-induced interleukin-6 (Il6) expression was repressed, in ATF3-deleted murine and human chondrocytes. Mechanistically, Atf3 deficiency decreased cytokine-induced Il6 transcription in chondrocytes through repressing NF-kB signalling by the attenuation of the phosphorylation status of IkB and p65. These findings suggest that Atf3 is implicated in the pathogenesis of OA through modulation of inflammatory cytokine expression in chondrocytes, and the feed-forward loop of inflammatory cytokines/NF-kB/Atf3 in chondrocytes may be a novel therapeutic target for the treatment for OA. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs.

Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine.

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 µM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

Upregulation of Runt-Related Transcription Factor-2 Through CCAAT Enhancer Binding Protein-ß Signaling Pathway in Microglial BV-2 Cells Exposed to ATP.

We have shown constitutive expression of the master regulator of osteoblastogenesis, runt-related transcription factor-2 (Runx2), by microglia cells outside bone. Here, we attempted to evaluate the pathological significance of Runx2 in microglial BV-2 cells exposed to ATP at a high concentration. Marked upregulation of Runx2 transcript and protein expression was seen in cells exposed to 1 mM ATP for a period longer than 30 min without inducing cytotoxicity. The Runx2 upregulation by ATP was prevented by extracellular and intracellular Ca(2+) chelators, while thapsigargin upregulated Runx2 expression alone without affecting the upregulation by ATP. A calmodulin antagonist prevented the upregulation by ATP, with calcineurin inhibitors being ineffective. Although ATP markedly increased nuclear levels of nuclear factor of activated T cell-2 (NFAT2), Runx2 promoter activity was not simulated by the introduction of either NFAT1 or NFAT2, but facilitated by that of CCAAT enhancer binding protein-α (C/EBPα), C/EBPß and nuclear factor (erythroid-derived 2)-like-2 (Nrf2). Exposure to ATP up-regulated C/EBPß and Nrf2, but not C/EBPα, expression, in addition to increasing nuclear levels of respective corresponding proteins. Runx2 upregulation by ATP was deteriorated by knockdown of C/EBPß but not by that of Nrf2, however, while exposure to ATP up-regulated matrix metalloproteinase-13 (Mmp13) expression in a Runx2-dependent manner. Overexpression of Runx2 up-regulated Mmp13 expression with promoted incorporation of fluorescent beads into BV-2 cells without ATP. These results suggest that extracellular ATP up-regulates Runx2 expression through activation of the C/EBPß signaling in a calmodulin-dependent manner to play a pivotal role in phagocytosis in microglial BV-2 cells.

Daily intake of ß-cryptoxanthin prevents bone loss by preferential disturbance of osteoclastic activation in ovariectomized mice.

Although ß-cryptoxanthin, a xanthophyll carotenoid, has been shown to exert an anabolic effect on bone calcification, little attention has been paid thus far to the precise mechanism of bone remodeling. Daily oral administration of ß-cryptoxanthin significantly inhibited osteoclastic activation as well as reduction of bone volume in ovariectomized mice. In vitro studies revealed that ß-cryptoxanthin inhibited differentiation and maturation of osteoclasts by repression of the nuclear factor-κB-dependent transcriptional pathway. Our results suggest that supplementation with ß-cryptoxanthin would be beneficial for prophylaxis and for therapy of metabolic bone diseases associated with abnormal osteoclast activation.

Daily oral intake of theanine prevents the decline of 5-bromo-2'-deoxyuridine incorporation in hippocampal dentate gyrus with concomitant alleviation of behavioral abnormalities in adult mice with severe traumatic stress.

Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2'-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress.

[Glutamic acid as a universal extracellular signal].

The prevailing view is that both glutamic (Glu) and gamma-aminobutyric (GABA) acids play a role as an amino acid neurotransmitter released from neurons. However, little attention has been paid to the possible expression and functionality of signaling machineries required for amino acidergic neurotransmission in cells other than central neurons. In line with our first demonstration of the presence of Glu receptors outside the brain, in this review I will outline our recent findings accumulated since then on the physiological and pathological significance of neuronal amino acids as an extracellular signal essential for homeostasis in a variety of phenotypic cells. In undifferentiated neural progenitor cells, for instance, functional expression is seen with different signaling machineries used for glutamatergic and GABAergic neurotransmission in neurons. Moreover, Glu plays a role in mechanisms underlying suppression of proliferation for self-replication in undifferentiated mesenchymal stem cells. There is more accumulating evidence for neuronal amino acids playing a role as an extracellular autocrine or paracrine signal commonly used in different phenotypic cells. Evaluation of drugs currently used could be thus beneficial for the efficient prophylaxis and/or the therapy of a variety of diseases relevant to disturbance of amino acid signaling in diverse organs.

Repression of adipogenesis through promotion of Wnt/ß-catenin signaling by TIS7 up-regulated in adipocytes under hypoxia.

Although tetradecanoyl phorbol acetate induced sequence-7 (TIS7) has been identified as a co-activator/repressor of gene transcription in different eukaryotic cells, little attention has been paid to the functionality of TIS7 in adipocytes. Here, we evaluated the possible role of TIS7 in mechanisms underlying the regulation of adipogenesis. TIS7 expression was preferentially up-regulated in white adipose tissues (WAT) of obesity model mice as well as in pre-adipocytic 3T3-L1 cells cultured under hypoxic conditions. TIS7 promoter activity was selectively enhanced by activating transcription factor-6 (ATF6) among different transcription factors tested, while induction of TIS7 by hypoxic stress was markedly prevented by knockdown of ATF6 by shRNA in 3T3-L1 cells. Overexpression of TIS7 markedly inhibited Oil Red O staining and expression of particular adipogenic genes in 3T3-L1 cells. TIS7 synergistically promoted gene transactivation mediated by Wingless-type mouse mammary tumor virus integration site family (Wnt)/ß-catenin, while blockade of the Wnt/ß-catenin pathway by a dominant negative form of T-cell factor-4 (DN-TCF4) markedly prevented the inhibition of adipogenesis in 3T3-L1 cells with TIS7 overexpression. TIS7 predominantly interacted with ß-catenin in the nucleus of WAT in the genetically obese ob/ob mice as well as in 3T3-L1 cells cultured under hypoxic conditions. Both knockdown of TIS7 by shRNA and introduction of DN-TCF4 similarly reversed the hypoxia-induced inhibition of adipogenic gene expression in 3T3-L1 cells. These findings suggest that TIS7 could play a pivotal role in adipogenesis through interacting with ß-catenin to promote the canonical Wnt signaling in pre-adipocytes under hypoxic stress such as obesity.

Nuclear factor-κB is a common upstream signal for growth differentiation factor-5 expression in brown adipocytes exposed to pro-inflammatory cytokines and palmitate.

We have previously demonstrated that genetic and acquired obesity similarly led to drastic upregulation in brown adipose tissue (BAT), rather than white adipose tissue, of expression of both mRNA and corresponding protein for the bone morphogenic protein/growth differentiation factor (GDF) member GDF5 capable of promoting brown adipogenesis. In this study, we evaluated expression profiles of GDF5 in cultured murine brown pre-adipocytes exposed to pro-inflammatory cytokines and free fatty acids (FFAs), which are all shown to play a role in the pathogenesis of obesity. Both interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were effective in up-regulating GDF5 expression in a concentration-dependent manner, while similar upregulation was seen in cells exposed to the saturated FFA palmitate, but not to the unsaturated FFA oleate. In silico analysis revealed existence of the putative nuclear factor-κB (NF-κB) binding site in the 5'-flanking region of mouse GDF5, whereas introduction of NF-κB subunits drastically facilitated both promoter activity and expression of GDF5 in brown pre-adipocytes. Chromatin immunoprecipitation analysis confirmed significant facilitation of the recruitment of NF-κB to the GDF5 promoter in lysed extracts of BAT from leptin-deficient ob/ob obese mice. Upregulation o GDF5 expression was invariably inhibited by an NF-κB inhibitor in cultured brown pre-adipocytes exposed to IL-1ß, TNF-α and palmitate. These results suggest that obesity leads to upregulation of GDF5 expression responsible for the promotion of brown adipogenesis through a mechanism relevant to activation of the NF-κB pathway in response to particular pro-inflammatory cytokines and/or saturated FFAs in BAT.