Epidermal peroxisome proliferator-activated receptor gamma as a target for ultraviolet B radiation

Ultraviolet B radiation (UVB) is a pro-oxidative stressor with profound effects on skin in part through its ability to stimulate cytokine production. Peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to regulate inflammatory processes and cytokine release in various cell types. Since the oxidized glycerophospholipid 1-hexadecyl-2-azelaoyl glycerophosphocholine (azPC) has been shown to be a potent PPAR gamma agonist, this study was designed to assess whether the PPAR gamma system is a target for UVB irradiation and involved in UVB-induced inflammation in epidermal cells. The present studies demonstrated the presence of PPAR gamma mRNA and functional protein in human keratinocytes and epithelial cell lines HaCaT, KB, and A431. The treatment of epidermal cells with the PPAR gamma-specific agonist ciglitazone or azPC augmented cyclooxygenase-2 expression and enzyme activity induced by phorbol 12-myristate-13-acetate or interleukin-1 beta. Lipid extracts from the cell homogenate of UVB-irradiated, but not control, cells contained a PPAR gamma-agonistic activity identified by reporter assay, and this activity up-regulated cyclooxygenase-2 expression induced by phorbol 12-myristate-13-acetate. Subjecting purified 1-hexadecyl-2-arachidonoyl-glycerophosphocholine to UVB irradiation generated a PPAR gamma-agonistic activity, among which the specific PPAR gamma agonist azPC was identified by mass spectrometry. These findings suggested that UVB-generated PPAR gamma-agonistic activity was due to the free radical mediated non-enzymatic cleavage of endogenous glycerophosphocholines. Treatment with the specific PPAR gamma antagonist GW9662 or expression of a dominant-negative PPAR gamma mutant in KB cells inhibited UVB-induced epidermal cell prostaglandin E(2) production. These findings suggested that UVB-generated PPAR gamma activity is necessary for the optimal production of epidermal prostaglandins. These studies demonstrated that epithelial cells contain a functional PPAR gamma system, and this system is a target for UVB through the production of novel oxidatively modified endogenous phospholipids.     

The liver X receptor ligand T0901317 down-regulates APOA5 gene expression through activation of SREBP-1c

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.     

Up-regulation of vascular endothelial growth factor C in breast cancer cells by heregulin-beta 1. A critical role of p38/nuclear factor-kappa B signaling pathway

Vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis that recently has been strongly implicated in the tumor metastasis process. In this study, we identified that HRG-beta 1 stimulated up-regulation of VEGF-C mRNA and protein of human breast cancer cells in a dosage- and time-dependent manner and that this up-regulation was de novo RNA synthesis-dependent. The HRG-beta 1-induced increase in VEGF-C expression was effectively reduced by treatment with Herceptin, an antibody specifically against HER2. Also, when HER2 was overexpressed in MCF-7 cells that resulted in an evident increase in the VEGF-C level, suggesting an essential role of HER2 in mediating VEGF-C up-regulation by HRG-beta 1. NF-kappa B has been shown to be probably involved in interleukin-1 beta- or tumor necrosis factor-alpha-induced VEGF-C mRNA expression in human fibroblasts. Here we found that HRG-beta 1 could stimulate NF-kappa B nuclear translocation and DNA-binding activity via the I kappa B alpha phosphorylation-degradation mechanism. Blockage of the NF-kappa B activation cascade caused a complete inhibition of the HRG-beta 1-induced elevation of VEGF-C. In promoter-reporter assay, the luciferase activities of the reporter constructs, including the putative NF-kappa B site deleted and mutated form were significantly reduced after HRG-beta 1 treatment as compared with the 1.5-kb VEGF-C promoter. Although investigating the upstream kinase pathway(s) involved in HRG-beta 1-elicited NF-kappa B activation and VEGF-C up-regulation, we found that HRG-beta1 could activate extracellular signal-regulated protein kinase 1/2, phosphatidylinositol 3'-kinase, and p38 mitogen-activated protein kinase (MAPK) in MCF-7. However, only SB203580 (a specific inhibitor of p38 MAPK), not PD98059 nor LY294002, blocked the up-regulation of VEGF-C by HRG-beta 1. A similar inhibition in VEGF-C expression was obtained by cell transfection with dominant-negative p38 (p38AF). Interestingly, the HRG-beta 1-induced NF-kappa B activation cascade was also effectively blocked by SB203580 treatment or p38AF transfection. Our data thus suggests that HRG-beta 1 stimulated a NF-kappa B-dependent up-regulation of VEGF-C through the p38 MAPK signaling pathway in human breast cancer cells.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis

TRAIL (Apo2 ligand) is a member of the tumor necrosis factor (TNF) family of cytokines that induces apoptosis. Because TRAIL preferentially kills tumor cells, sparing normal tissues, interest has emerged in applying this biological factor for cancer therapy in humans. However, not all tumors respond to TRAIL, raising questions about resistance mechanisms. We demonstrate here that a variety of natural and synthetic ligands of peroxisome proliferator-activated receptor-gamma (PPAR gamma) sensitize tumor but not normal cells to apoptosis induction by TRAIL. PPAR gamma ligands selectively reduce levels of FLIP, an apoptosis-suppressing protein that blocks early events in TRAIL/TNF family death receptor signaling. Both PPAR gamma agonists and antagonists displayed these effects, regardless of the levels of PPAR gamma expression and even in the presence of a PPAR gamma dominant-negative mutant, indicating a PPAR gamma-independent mechanism. Reductions in FLIP and sensitization to TRAIL-induced apoptosis were also not correlated with NF-kappa B, further suggesting a novel mechanism. PPAR gamma modulators induced ubiquitination and proteasome-dependent degradation of FLIP, without concomitant reductions in FLIP mRNA. The findings suggest the existence of a pharmacologically regulated novel target of this class of drugs that controls FLIP protein turnover, and raise the possibility of combining PPAR gamma modulators with TRAIL for more efficacious elimination of tumor cells through apoptosis.     

马齿苋多糖对溃疡性结肠炎大鼠肠组织IL-6/STAT3及NF-κB的影响

目的：采用TNBS （2,4,6-三硝基苯磺酸）复制溃疡性结肠炎大鼠模型,探索马齿苋多糖对溃疡性结肠炎大鼠肠组织IL6/STAT3及NF-κB的影响,明确IL-6/STAT3信号通路与慢性炎症性肠病发病的关系,为慢性溃疡性结肠炎的治疗寻找新靶点。方法：将40只SD大鼠随机分为对照组、模型组、美沙拉嗪组和马齿苋组（n=10）。采用TNBS诱导复制结肠炎模型,造模成功后第3天开始灌胃给药：美沙拉嗪组剂量为每次10 mg/kg,每日1次,连续3周;马齿苋组给予马齿苋多糖,每次10 ml/kg,每日1次,连续3周;模型组和对照组大鼠给予等体积生理盐水灌胃,每日1次,连续3周。收集大鼠结肠内容物称重,干燥后再次称重,取结肠组织作病理切片。采用ELISA试剂盒检测血清IL-6、IL-1β、TNF-α和核转录因子-kappa B （NF-κB）含量;免疫组化染色法测定结肠髓过氧化物酶（MPO）;RT-PCR法检测信号转导和转录激活因子（STAT3）、IL-6的mRNA。结果：与模型组、美沙拉嗪组比较,马齿苋组大鼠排便状态明显改善,肠粘膜水肿减轻;血清IL-6、sIL-6Rα、gp130,肠组织MPO、NF-κB含量均降低（P<0.01）。与模型组比较,马齿苋组STAT3、IL-6mRNA的表达水平明显降低（P<0.01）。与对照组比较,上述指标无显著性差异（P>0.05）。结论：马齿苋多糖通过降低大鼠血清IL-6、sIL-6Rα、gp130含量及肠组织MPO、NF-κB水平,减轻sIL-6Rα与IL-6形成复合物所致的炎症反应;经IL-6/STAT3信号通路下调大鼠肠组织STAT3和IL-6的mRNA水平,从而抑制炎症的发生。     

PPAR gamma ligand troglitazone lowers cholesterol synthesis in HepG2 and Caco-2 cells via a reduced concentration of nuclear SREBP-2

Cholesterol synthesis in animal cells is regulated by sterol regulatory element-binding protein (SREBP)-2. The objective of this study was to investigate whether activation of peroxisome proliferator-activatedreceptor (PPAR)-gamma influences the SREBP-2 dependent cholesterol synthesis in liver and intestinal cells. Therefore, HepG2 and Caco-2 cells were incubated with and without 10 or 30 microM of troglitazone, a synthetic PPAR gamma agonist, for 4 hrs. Incubation with 10 or 30 microM of troglitazone caused a significant, dose-dependent reduction of cholesterol synthesis in both HepG2 and Caco-2 cells (P < 0.05). HepG2 and Caco-2 cells incubated with 10 or 30 microM of troglitazone had also lower mRNA concentrations and lower nuclear protein concentrations of SREBP-2 than untreated control cells (P < 0.05). mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase and LDL receptor were also reduced in HepG2 and Caco-2 cells treated with 30 microM of troglitazone compared to control cells (P < 0.05). In conclusion, this study shows that PPAR gamma activation by troglitazone lowers the cholesterol synthesis in HepG2 and Caco-2 cells by reducing the concentration of nuclear SREBP-2 and successive downregulation of its target genes involved in cholesterol synthesis.     

Impaired expression of PPAR gamma protein contributes to the exaggerated growth of vascular smooth muscle cells in spontaneously hypertensive rats

Peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor family, has been implicated in the regulation of vascular smooth muscle cell (VSMC) growth; however, the underlying mechanisms are still not fully understood. We hypothesized that PPAR gamma functional deficiency may contribute to the enhanced proliferation of VSMC associated with hypertension in spontaneously hypertensive rats (SHR). We observed that PPAR gamma mRNA level in SHR VSMC was 3 approximately 4 fold higher than that from Wistar-Kyoto rats (WKY), but the protein expression levels of PPAR gamma are significantly lower in SHR than WKY VSMC, suggesting an impaired control of PPAR gamma protein expression in SHR VSMC. The deficiency of PPAR gamma protein expression in SHR VSMC was demonstrated by PPAR gamma reporter gene assays. Furthermore, the exaggerated growth of SHR VSMC was markedly attenuated by adenoviral PPAR gamma overexpression. Taken together, our results provided the first direct evidence that impaired expression of PPAR gamma protein contributes to the exaggerated growth of SHR VSMC.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.