Takashi Nagaya

Nuclear receptor corepressors activate rather than suppress basal transcription of genes that are negatively regulated by thyroid hormone.

A group of transcriptional cofactors referred to as corepressors (CoRs) were recently shown to play a central role in basal silencing of genes that contain positive triiodothyronine (T3) response elements. In a reciprocal manner, negatively regulated genes are stimulated by unliganded thyroid hormone receptor (TR) and repressed upon the addition of T3. We used a TR beta mutant, called P214R, which fails to interact with CoRs, to examine whether CoRs also play a role in the control of genes that are negatively regulated in response to T3. In studies of three negatively regulated genes (the pituitary thyroid-stimulating hormone alpha-subunit [TSH alpha], TSH beta, and hypothalamic thyrotropin-releasing hormone [TRH] genes), stimulation of basal promoter activity by unliganded TR beta was impaired by introducing the P214R CoR mutation. Coexpression of each of the CoRs SMRT (silencing mediator for retinoid receptors and TRs) and NCoR (nuclear receptor CoR) enhanced basal stimulation of the negatively regulated promoters in a TR-dependent manner, but this effect was not seen with the P214R TR mutant. The mechanism of CoR effects on negatively regulated promoters was explored further with a series of GAL4-TR chimeric receptors and mutants that allowed TR effects to be assessed independently of receptor interactions with DNA. These experiments revealed that, like the negative regulation of genes by wild-type TR, basal activation occurred with GAL4-TR, but not with the GAL4-P214R mutant, and was reversed by the addition of T3. These results suggest that TR interactions with negatively regulated genes may be driven through protein-protein interactions. We conclude that a subset of negatively regulated genes are controlled by a novel mechanism that involves TR-mediated recruitment and basal activation by SMRT and NCoR. Addition of T3 reverses basal activation, perhaps by dissociation of CoRs.

Inhibition of NF-κB Activity Decreases the VEGF mRNA Expression in MDA-MB-231 Breast Cancer Cells

VEGF (vascular endothelial growth factor) secreted from tumor cells including breast cancer serves as a potent angiogenic factor which favors tumor growth and metastasis. Indeed, a higher concentration of serum VEGF has been shown to associate with a poorer prognosis in patients with breast cancer. On the other hand, constitutive expression of a transcription factor, NF-κB was correlated with progression and metastasis in a number of human breast cancers, suggesting a possible regulation of VEGF expression by NF-κB. We thus investigated the relationship between the expression of VEGF and constitutive NF-κB activity in three breast cancer cell lines, MCF-7, T47D, and MDA-MB-231. The basal levels of VEGF mRNA expression correlated with those of nuclear NF-κB activity in these cell lines. The highest NF-κB activity in MDA-MB-231 cells was associated with the highest expression of VEGF mRNA, while the activity and the mRNA levels were moderate in MCF cells and the lowest in T47D cells. In MDA-MB-231 cells, inhibition of NF-κB by adenovirus-mediated expression of a dominant negative NF-κB or by a proteasome inhibitor, MG132, decreased the VEGF mRNA. These results suggest that NF-κB is involved in the upregulation of VEGF mRNA and inhibition of the activity could be a new approach for the treatment of breast cancer by preventing angiogenesis.

Culture in Vector-Averaged Gravity Under Clinostat Rotation Results in Apoptosis of Osteoblastic ROS 17/2.8 Cells

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector‐averaged gravity environment. We then evaluated the responses of osteoblast‐like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6–24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell‐surface integrin β1 and disorganization of actin cytoskeleton. These results suggest that vector‐averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.

Overexpression of calpastatin by gene transfer prevents troponin I degradation and ameliorates contractile dysfunction in rat hearts subjected to ischemia/reperfusion

Calpain is a Ca(2+)-activated neutral protease that supposedly plays a key role in myocardial dysfunction following ischemia/reperfusion, by degrading certain proteins involved in the contraction mechanism. It is possible that overexpression of calpastatin, an endogenous calpain inhibitor, lessens contractile dysfunction in the heart after reperfusion by preventing cardiac troponin I (TnI) degradation. This claim is tested by overexpression of human calpastatin (hCS) in rat hearts ex vivo using an adenovirus vector; the hearts were transplanted heterotopically into the abdomens of recipient rats to allow expression of hCS. On the fourth day after surgery, the hearts were excised and perfused in vitro to study their recovery from 30 min of global ischemia, which was followed by 60 min of reperfusion. The peak recovery of the left ventricular developed pressure (LVDP), and the values of its first derivative (max dP/dt, min dP/dt) in the hCS-overexpressed hearts were 88.9 +/- 4.8%, 90.8 +/- 9.2% and 106.4 +/- 9.8%, respectively; these values were all significantly greater than in the control hearts transfected with LacZ alone (51.4 +/- 6.9%, 52.6 +/- 8.1% and 54.7 +/- 6.6%, P < 0.05). In western blot analysis of ventricular myocardial samples (at 60-min reperfusion) using a monoclonal anti-TnI antibody, two bands corresponding to intact TnI (30 kDa) and TnI fragments (27 kDa) were distinguished. The fraction of 27-kDa TnI (percent of total TnI immunoreactivity) in hCS-overexpressed hearts was significantly less than the controls (5.7 +/- 2.7% vs. 18.1 +/- 3.2%, P < 0.05), implying a protective action of hCS against TnI degradation. These results suggest that adenovirus-mediated overexpression of hCS in the heart could be a novel biological means to minimize myocardial stunning by ischemia/reperfusion.

Activation of Transcriptionally Active Nuclear Factor-κB by Tumor Necrosis Factor-α and Its Inhibition by Antioxidants in Rat Thyroid FRTL-5 Cells1

Tumor necrosis factor-α (TNF-α) exerts pleiotropic effects on thyroid follicular cells. However, the intracellular signaling pathway for the TNF-α action has not been well elucidated. The present study examined the effects of TNF-α on the activation of nuclear factor-κ B (NF-κB) and on the expression of interleukin (IL)-6 gene in rat thyroid FRTL-5 cells. The treatment of the cells with TNF-α resulted in the nuclear translocation of p65-p50 heterodimer as well as p50-p50 homodimer NF-κBs. The treatment with the antioxidants 20 mm N-acetyl-L-cysteine (NAC) and 10 μm pyrrolidine dithiocarbamate (PDTC) inhibited the TNF-α-dependent activation of p65-p50 heterodimer but not the p50-p50 homodimer, indicating that generation of oxidants is required for the activation of the heterodimer NF-κB. When the plasmid containing the multimerized NF-κB sites upstream of a luciferase reporter gene was transfected into FRTL-5 cells, the treatment with NAC or PDTC prevented the TNF-α-dependent increase in the luciferase act...

Inhibitory effects of cyclosporin A on calcium mobilization-dependent interleukin-8 expression and invasive potential of human glioblastoma U251MG cells.

Interleukin (IL)-8 produced from glioblastoma is suggested to contribute to its own proliferation and progression. Since various external stimuli have been shown to increase intracellular Ca2+ in glioma cells, we investigated Ca2+ mobilization-dependent IL-8 expression and effect of cyclosporin A (CsA), an inhibitor of calcineurin (Cn), on the expression and invasive potential of human glioblastoma U251MG cells. Combined treatment with Ca2+-ionophore and phorbol-myristate-acetate (A23187/PMA) increased IL-8 mRNA and protein levels. This increase was suppressed by CsA and by another Cn inhibitor FK506. Luciferase reporter gene assay and electrophoretic mobility shift assay revealed that activation of p65-containing nuclear factor-κB was essential for A23187/PMA-dependent activation of IL-8 promoter. CsA suppressed the promoter activity by attenuating IκB-α degradation. U251MG cells expressed IL-8 receptors CXCR-1 and -2, and Matrigel invasion assay revealed that CsA attenuated A23187/PMA-dependent stimulation of invasive potential, probably by inhibiting IL-8 production. In addition, IL-8-dependent proliferation was also suppressed by CsA. Taken together, these results demonstrate the novel inhibitory effects of CsA on glioblastoma cell functions, suggesting CsA as a potential therapeutic adjuvant for glioma treatment.

Expression of PPARγ and Its Ligand-dependent Growth Inhibition in Human Brain Tumor Cell Lines

Peroxisome proliferator‐activated receptor y (PPARγ) belongs to a superfamily of thyroid/steroid hormone receptors and regulates transcription of their target genes in a ligand‐dependent manner. Recently, PPARγ was reported to be expressed in several cell lines derived from breast, colon, stomach and lung cancers. Activation of PPARγ by its ligand inhibits the growth of these tumor cells, suggesting that PPARγ ligand is a potential anti‐cancer agent in PPARγ‐expressing tumors. However, its expression in brain tumors has not been studied. We thus studied the expression in glioma samples with different pathological stages from 20 patients. It was demonstrated that 95% of the glioma tissue expressed PPARγ mRNA. The results prompted us to study whether PPARγ ligand affects the growth of cell lines derived from brain tumors. The receptor expression was studied in 9 cell lines either derived from malignant glioma or neuroblastoma. The expression was detected in a glioma cell line SK‐MG‐1 and in a neuroblastoma cell line NB‐1. Addition of one of the PPARγ ligands, troglitazone, induced growth inhibition in both cell lines. Further analyses revealed that this growth inhibition is caused by a PPARγ‐mediated induction of apoptosis. These results suggest that PPARγ ligands could be a potential therapeutic agent for the treatment of the brain tumors expressing this receptor.

Inductions of immediate early genes (IEGs) and ref-1 by human chorionic gonadotropin in murine Leydig cell line (MA-10)

The effect of human chorionic gonadotropin (hCG) on the expression of immediate early genes (IEGs) including all members of fos and jun family, and c‐myc was studied using mouse Leydig cell line (MA‐10 cells) by Northern blot analyses. In addition, the induction of ref‐1 which enhances DNA binding of fos/jun proteins was also analyzed. HCG induced a rapid and transient expression of c‐fos, fosB, c‐jun, junB, junD and c‐myc with a peak at 30 min to 1h. In contrast, induction of fra‐1 mRNA was delayed with a peak at 3 hr. However, fra‐2 mRNA was immediately increased by hCG with a peak at 1 h. The ref‐1 mRNA was expressed before the stimulation and its level was not altered by hCG at least for 8 hr. The differential induction of IEGs and continuous expression of ref‐1 mRNA suggest an important role of their gene products on the regulation of Leydig cell function by hCG.

NGFI-B, c-fos, and c-jun mRNA Expression in Mouse Brain After Acute Carbon Monoxide Intoxication

The expression of immediate early genes (IEG) has been documented in the brain after various kinds of insults such as ischemia and hypoxia. To determine whether acute carbon monoxide intoxication (ACOI) might trigger IEG expression, adult ddY mice were subjected to carbon monoxide exposure at a rate of 30 mL/min for 35 seconds. The levels of NGFI-B, c-fos, and c-jun mRNA were determined by Northern blot analysis. A time-course study in the cerebral cortex indicated that the induction of NGFI-B, c-fos, and c-jun mRNA started as early as 15 minutes, reached a peak at 30 minutes, and returned to the basal level at 1 hour after the ACOI. In addition, the temporal feature of the induction of these IEG mRNA in the hippocampus was very similar to that in the cerebral cortex. Examination of brain regions at 30 minutes after the ACOI revealed a significant induction of NGFI-B mRNA in the cerebellum, thalamus-hypothalamus, brainstem, as well as in the cortex and hippocampus, but not in the striatum or olfactory bulb. Furthermore, the neuroanatomical distribution of c-fos mRNA at 30 minutes after the ACOI was very similar to that of the NGFI-B mRNA. The widespread distribution of these IEG in the brain, especially in the cerebellum and brainstem, indicates that the major cause for the triggering of IEG expression in the brain by the ACOI might be a diffuse hypoxia. These findings show for the first time the temporal and spatial expression of IEG in the brain after ACOI.

Thyrotropin modifies activation of nuclear factor kappaB by tumour necrosis factor alpha in rat thyroid cell line.

We have recently demonstrated that nuclear factor kappaB (NF-kappaB) mediates the tumour necrosis factor alpha (TNF-alpha)-dependent expression of the gene encoding interleukin 6 (IL-6) in rat thyroid FRTL-5 cells cultured in the presence of thyrotropin (TSH). In the present study we investigated how TSH is involved in the activation of NF-kappaB by TNF-alpha in the cells. Electrophoretic mobility-shift assay revealed that, in the absence of TSH, TNF-alpha activated a single protein-DNA complex containing the p50 subunit but not other NF-kappaB subunits such as p65. In contrast, two distinct protein-DNA complexes were activated in the presence of TSH: the faster-migrating complex contained only p50 subunit; the slower-migrating complex consisted of p65-p50 heterodimer. This TSH effect was mimicked by forskolin and thyroid-stimulating antibodies obtained from patients with Gravess disease, suggesting that an increase in intracellular cAMP is responsible for the induction of different NF-kappaBs by TNF-alpha. A transient transfection study with a luciferase reporter gene driven by multimerized NF-kappaB sites demonstrated that TNF-alpha increased the luciferase activities only in the presence of TSH, and that this increase was inhibited by the co-transfection of mutant p65, which prevented the function of wild-type p65 in a dominant-negative manner. Accordingly, TNF-alpha activated the expression of the IL-6 gene in the presence of TSH but not in its absence. Although the expression of the p105 gene, another known target for NF-kappaB, was increased by TNF-alpha in the absence of TSH, the presence of TSH further increased the mRNA level. Taken together, these observations indicate that the presence of TSH is crucial for the NF-kappaB-mediated actions of TNF-alpha on thyroid follicular cells.