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IL-2 gene expression in activated T-cells is initiated by chromatin remodeling at the IL-2 proximal promoter and conversion of a transcriptional repressor into a potent transcriptional activator. A purine-box regulator complex was purified from activated Jurkat T-cell nuclei based on sequence-specific DNA binding to the antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target DNA sequence in the proximal IL-2 promoter. ARRE DNA-binding subunits were identified as NF90, NF45 and systemic lupus erythematosis autoantigens, Ku80 and Ku70. Monoclonal antibodies to Ku80, Ku70 and NF90 specifically inhibit constitutive and inducible ARRE DNA-binding activity in Jurkat T-cells. Ku80, Ku70 and NF90 bind specifically to the IL-2 gene promoter in vivo, as demonstrated by chromatin immunoprecipitation. Activation of Jurkat T-cells and mouse primary spleen cells induces binding of Ku80 and NF90 to the IL-2 promoter in vivo, and decreases binding of Ku70 to the IL-2 promoter in vivo, and these dynamic changes are inhibited by immunosuppressants cyclosporin A and triptolide. Dynamic changes in binding of Ku80, Ku70 and NF90 to the IL-2 proximal promoter in vivo correlate with chromatin remodeling and transcriptional initiation in activated T-cells.

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

NF90 and splice variant NF110/ILF3/NFAR are double-stranded RNA-binding proteins that regulate gene expression. Mice with targeted disruption of NF90 were engineered. NF90(-/-) mice were born small and weak and succumbed to perinatal death within 12 h because of neuromuscular respiratory failure. Lung inflation and morphology were normal in NF90(-/-) mice. The diaphragm and other skeletal muscles in NF90(-/-) mice demonstrated disorganized arrangement and paucity of myofibers, evidence of myocyte degeneration and increased apoptosis. The expression of myogenic regulators, MyoD, myogenin, and p21WAF1/CIP1, was severely decreased in NF90(-/-) mice. These myogenic transcription factors and cell cycle inhibitors are regulated in part through post-transcriptional mRNA stabilization. Northwestern blotting revealed that NF90 is the principal and specific p21WAF1/CIP1 and MyoD 3'-untranslated region RNA-binding protein in developing skeletal muscles. NF90 regulates transcription factors and a cell cycle inhibitor essential for skeletal muscle differentiation and for survival.

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.

In 16HBE transformed human bronchial epithelial cells, histamine stimulated interleukin (IL)-8 mRNA and protein secretion, and this histamine stimulation was inhibited by the H1-receptor antagonist diphenhydramine (DPH), by the inhibitor of 5-lipoxygenase-activating protein (FLAP) MK-886, by the 5-lipoxygenase inhibitor Zileuton, and by dexamethasone. Histamine stimulated bronchial epithelial cell production of leukotriene B4(LTB4), and this production was inhibited by FLAP inhibitors MK-886 and L-655,238 and Zileuton. Histamine stimulated IL-8 luciferase reporter gene activity that was inhibited with DPH, dexamethasone, MK-886 and L-655,238, and Zileuton. The inhibition of IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was reversed with exogenous LTB4. There was increased IL-8 nuclear factor-κB (NF-κB) DNA-binding activity after histamine stimulation, and this was inhibited by DPH and MK-886. Cytoplasmic phospholipase A2mRNA levels were also potently induced by histamine. Thus histamine stimulation of bronchial epithelial cells involves binding at H1receptors, production of LTB4, activation of NF-κB and increased expression of IL-8.

Pneumonectomized rats injected with the alkaloid toxin, monocrotaline, develop progressive neointimal pulmonary vascular obliteration and pulmonary hypertension resulting in right ventricular failure and death. The antiproliferative immunosuppressant, triptolide, attenuates neointimal formation and pulmonary hypertension in this disease model (Faul JL, Nishimura T, Berry GJ, Benson GV, Pearl RG, and Kao PN. Am J Respir Crit Care Med 162: 2252–2258, 2000). Pneumonectomized rats, injected with monocrotaline on day 7, were killed at days 14, 21, 28, and 35 for measurements of physiology and gene expression patterns. These data were compared with pneumonectomized, monocrotaline-injected animals that received triptolide from day 5 to day 35. The hypothesis was tested that a group of functionally related genes would be significantly coexpressed during the development of disease and downregulated in response to treatment. Transcriptional analysis using total lung RNA was performed on replicate animals for each experimental time point with exploratory data analysis followed by statistical significance analysis. Marked, statistically significant increases in proteases (particularly derived from mast cells) were noted that parallel the development of vascular obliteration and pulmonary hypertension. Mast-cell-derived proteases may play a role in regulating the development of neointimal pulmonary vascular occlusion and pulmonary hypertension in response to injury.

Background— Pulmonary vascular injury by toxins can induce neointimal formation, pulmonary arterial hypertension (PAH), right ventricular failure, and death. We showed previously that simvastatin attenuates smooth muscle neointimal proliferation and pulmonary hypertension in pneumonectomized rats injected with the alkaloid toxin monocrotaline. The present study was undertaken to investigate the efficacy of simvastatin and its mechanism of reversing established neointimal vascular occlusion and pulmonary hypertension. Methods and Results— Pneumonectomized rats injected with monocrotaline at 4 weeks demonstrated severe PAH at 11 weeks (mean pulmonary artery pressure [mPAP]=42 versus 17 mm Hg in normal rats) and death by 15 weeks. When rats with severe PAH received simvastatin (2 mg · kg −1 · d −1 by gavage) from week 11, there was 100% survival and reversal of PAH after 2 weeks (mPAP=36 mm Hg) and 6 weeks (mPAP=24 mm Hg) of therapy. Simvastatin treatment reduced right ventricular hypertrophy and reduced proliferation and increased apoptosis of pathological smooth muscle cells in the neointima and medial walls of pulmonary arteries. Longitudinal transcriptional profiling revealed that simvastatin downregulated the inflammatory genes fos, jun, and tumor necrosis factor-α and upregulated the cell cycle inhibitor p27Kip1, endothelial nitric oxide synthase, and bone morphogenetic protein receptor type 1a. Conclusions— Simvastatin reverses pulmonary arterial neointimal formation and PAH after toxic injury.

Extracts of Tripterygium wilfordii hook. f. (leigong teng, Thundergod vine) are effective in traditional Chinese medicine for treatment of immune inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus, nephritis and asthma. Characterisation of the terpenoids present in extracts of Tripterygium identified triptolide, a diterpenoid triepoxide, as responsible for most of the immunosuppressive, anti-inflammatory and antiproliferative effects observed in vitro. Triptolide inhibits lymphocyte activation and T-cell expression of interleukin-2 at the level of transcription. In all cell types examined, triptolide inhibits nuclear factor-kappaB transcriptional activation at a unique step in the nucleus after binding to DNA. Further characterisation of the molecular mechanisms of triptolide action will serve to elucidate pathways of immune system regulation.

Hypertensive pulmonary vascular disease is characterized by abnor- prostanoids (7), which dilate reactive blood vessels and supmal proliferation of vascular endothelial and smooth muscle cells, press the growth of smooth muscle cells (8). Additional antileading to occlusion of pulmonary arterioles, pulmonary hyperten- proliferative strategies for treatment of vascular occlusive sion, right ventricular failure, and death. Compounds with antipro- diseases are warranted (9). liferative effects on vascular endothelial and smooth muscle cells, Our group has previously demonstrated that antiproliferasuch as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reduc- tive drugs with no acute vasodilator properties effectively tase inhibitors, may prevent the development of experimental hy- attenuate vascular remodeling with neointimal formation, pertensive pulmonary vascular disease. Pneumonectomized rats in- pulmonary arterial hypertension, and right ventricular hyperjected with monocrotaline at 7 days develop severe hypertensive trophy in pneumonectomized, monocrotaline-injected rats (10, pulmonary vascular disease with neointimal formation. Rats were 11). The combination of pneumonectomy with monocrotaline randomized to receive either vehicle or treatment with the HMGCoA reductase inhibitor simvastatin (2 mg/kg per day). By Day injection results in more severe pulmonary arterial hyperten35, rats that received vehicle had higher mean pulmonary arterial sion (mean pulmonary arterial blood pressure [Ppa] 45 mm pressures (53 2 mm Hg) and right ventricular hypertrophy (right Hg) (10–12) than monocrotaline alone (Ppa 32 mm Hg) ventricle/[left ventricle plus septum] [RV/LVS] 0.78 0.09) (13–15). Importantly, only the combination of pneumonecthan rats in Group PMS5–35 that received simvastatin from Day 5 to tomy with monocrotaline injection produces neointimal for35 (mean pulmonary arterial pressure 27 3 mm Hg, RV/LVS mation and vascular obliteration of small pulmonary arteri0.34 0.08; p 0.001). Pulmonary vascular remodeling with neointi- oles that reproduces many of the pathological features of mal formation consisting of vascular smooth muscle cells was more human PPH (2, 9). severe in vehicle-treated rats (vascular occlusion score, 1.98 0.02) It is increasingly recognized that treatment with 3-hydroxthan in Group PMS5–35 (vascular occlusion score, 0.59 0.46; p ymethyl-3-methylglutaryl-coenzyme A (HMG-CoA) reduc

Triptolide (PG490, 97% pure) is a diterpenoid triepoxide with potent anti-inflammatory and immunosuppressive effects in transformed human bronchial epithelial cells and T cells (Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC-H, and Kao PN. J Biol Chem 274: 13443–13450, 1999). Triptolide, with an IC50of ∼20–50 ng/ml, inhibits normal and transformed human bronchial epithelial cell expression of interleukin (IL)-6 and IL-8 stimulated by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-α, or IL-1β. Nuclear runoff and luciferase reporter gene assays demonstrate that triptolide inhibits IL-8 transcription. Triptolide also inhibits the transcriptional activation, but not the DNA binding, of nuclear factor-κB. A cDNA array and clustering algorithm analysis reveals that triptolide inhibits expression of the PMA-induced genes tumor necrosis factor-α, IL-8, macrophage inflammatory protein-2α, intercellular adhesion molecule-1, integrin β6, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, GATA-3, fra-1, and NF45. Triptolide also inhibits constitutively expressed cell cycle regulators and survival genes cyclins D1, B1, and A1, cdc-25, bcl-x, and c-jun. Thus anti-inflammatory, antiproliferative, and proapoptotic properties of triptolide are associated with inhibition of nuclear factor-κB signaling and inhibition of genes known to regulate cell cycle progression and survival.

PG490 (triptolide) is a diterpene triepoxide with potent immunosuppressive and antiinflammatory properties. PG490 inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and antibody to CD3 (IC50 of 10 ng/ml), and with PMA and ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells, PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits the induction of DNA binding activity at the purine-box/antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target sequence but not at the NF-kappaB site. PG490 can completely inhibit transcriptional activation at the purine-box/ARRE/NF-AT and NF-kappaB target DNA sequences triggered by all stimuli examined (PMA, PMA/Iono, tumor necrosis factor-alpha). PG490 also inhibits PMA-stimulated activation of a chimeric transcription factor in which the C-terminal TA1 transactivation domain of NF-kappaB p65 is fused to the DNA binding domain of GAL4. In 16HBE human bronchial epithelial cells, IL-8 expression is regulated predominantly by NF-kappaB, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene expression differs from cyclosporin A and involves nuclear inhibition of transcriptional activation of NF-kappaB and the purine-box regulator operating at the ARRE/NF-AT site at a step after specific DNA binding.

Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L276–L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136–2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.

Human airway epithelial cells (AEC) produce the T cell growth factor interleukin (IL)-2 that likely modulates the T cell lung inflammatory response. IL-2 mRNA from cultured AEC and from Jurkat T cells was analyzed by reverse transcription-polymerase chain reaction and Northern hybridization. IL-2 mRNA is present constitutively in AEC and is enhanced twofold after stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml) + histamine (2 mM). Normal human AEC secrete IL-2 at rest (7 pg/ml), and IL-2 secretion is increased threefold after stimulation with PMA + histamine; this increase is inhibited by dexamethasone and diphenhydramine. Transcriptional regulation of IL-2 was investigated with a transgenic human AEC line, 16HBE/IL-2 luciferase; there is constitutive IL-2 transcription at rest, and IL-2 transcription is enhanced 8-fold by PMA and 25-fold by PMA + histamine. IL-2 regulation differs fundamentally between AEC and Jurkat T cells. AEC IL-2 likely promotes local proliferation of T cells and may contribute to pathological airway inflammation in asthma.