Long non-coding RNAs TUG1 and MEG3 in patients with type 2 diabetes and their association with endoplasmic reticulum stress markers.

BACKGROUND: Long non-coding RNAs (lncRNAs), including taurine upregulated gene 1 (TUG1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and maternally expressed 3 (MEG3) play a regulatory role in endoplasmic reticulum (ER) stress. The present study aimed to investigate the expression of these lncRNAs in patients with type 2 diabetes and their association with biochemical and ER stress parameters. MATERIALS AND METHODS: Participants included 57 patients with diabetes and 32 healthy individuals. Real-time PCR was performed to assess MALAT1, TUG1, MEG3, ATF4, and CHOP gene expression in peripheral blood mononuclear cells. Plasma GRP78, advanced glycation end products (AGEs), and insulin were measured using enzyme-linked immunosorbent assay (ELISA), and insulin resistance (IR) was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: The expression of TUG1, MEG3, ATF4, and CHOP genes was significantly increased in the patients with diabetes compared to healthy individuals. MALAT1 gene expression was also higher in patients group; although it did not reach significant levels. TUG1 and MEG3 expression revealed significant positive correlations with the indices of glycemic control, including FBS, HbA1c, HOMA-IR, and AGEs, as well as markers of ER stress. MALAT1 expression was also positively correlated with ATF4 and AGEs. CONCLUSION: The expression levels of TUG1 and MEG3 lncRNAs were increased in patients with diabetes and were associated with glycemic control and components of ER stress. Thus, these lncRNAs might be considered appropriate markers to identify ER stress due to hyperglycemia.

Circulating TRB3 and GRP78 levels in type 2 diabetes patients: crosstalk between glucose homeostasis and endoplasmic reticulum stress.

PURPOSE: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus (T2DM) and insulin resistance. Tribbles homolog 3 (TRB3) is a pseudokinase upregulated by ER stress and hyperglycemia. Glucose-regulated protein 78 (GRP78) is an ER stress protein that is overexpressed under ER stress conditions. The current study aimed to investigate serum levels of TRB3 and GRP78, as an ER stress marker, in T2DM patients and their correlations with the metabolic profile. METHODS: Fifty-seven patients with type 2 diabetes and 23 healthy control subjects were evaluated for serum concentrations of TRB3, GRP78, and AGEs by enzyme-linked immunosorbent assay (ELISA). Fasting plasma glucose (FPG), HbA1c, lipid profile, TNF-α and insulin were also measured, and insulin resistance was calculated using a homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: Serum concentrations of TRB3, GRP78, AGEs, and TNF-α were significantly higher in T2DM patients compared to the healthy controls. Moreover, a statistically significant positive correlation was observed between plasma concentrations of TRB3 and FPG, HbA1c, HOMA-IR, and AGE. GRP78 levels were positively correlated with HbA1c and AGEs. There was also a positive correlation between GRP78 and TRB3. AGEs levels were positively correlated with the levels of FPG, HbA1c, HOMA-IR, and TNF-α. CONCLUSION: The current findings suggest that TRB3 and GRP78 may contribute to the pathogenesis of T2DM and might be considered as a therapeutic targets for the treatment of this disease.

Peroxisome proliferator-activated receptor gamma expression in peripheral blood mononuclear cells and angiopoietin-like protein 4 levels in obese children and adolescents.

PURPOSE: The peroxisome proliferator-activated receptor γ (PPARγ) is highly expressed in adipose tissue and functions as transcriptional regulator of metabolism and adipocyte differentiation. Angiopoietin-like protein 4 (ANGPTL4), a central player in various aspects of energy homoeostasis, is induced by PPARγ. The aim of this study was to evaluate ANGPTL4 plasma levels and PPARγ gene expression in peripheral blood mononuclear cells (PBMCs) of children and adolescents with obesity and their association with metabolic parameters. METHODS: Seventy children and adolescents (35 obese and 35 age- and gender-matched control subjects), were selected. PBMCs were separated and their total RNA was extracted. After cDNA synthesis, PPARG gene expression was analyzed by real-time PCR. Relative differences in gene expression were calculated by ΔCt method using ß-actin as a normalizer. Serum ANGPTL4 and insulin were measured using ELISA, and insulin resistance (IR) was calculated by the homeostatic model assessment of insulin resistance (HOMA-IR). Fasting plasma glucose (FPG), triglyceride, total cholesterol, LDL-C and HDL-C were also measured. RESULTS: The expression of the PPARG gene as well as the plasma ANGPTL4 levels were significantly diminished in obese subjects as compared to control ones. However, they were not significantly different in obese children with IR compared to obese children without IR or in those with or without metabolic syndrome. A significant positive correlation was found between PPARγ and ANGPTL4 (r = 0.364, p = 0.002). PPARγ expression levels were also significantly correlated with FPG (r = -0.35, p = 0.003). CONCLUSION: PPARγ is decreased in childhood obesity and may be responsible for diminished ANGPTL4 levels.

THE ASSOCIATION OF PLASMA LEVELS OF miR-34a AND miR-149 WITH OBESITY AND INSULIN RESISTANCE IN OBESE CHILDREN AND ADOLESCENTS.

CONTEXT: MicroRNAs (miRNAs) are short noncoding RNAs involved in posttranscriptional regulation of gene expression that influence various cellular functions including glucose and lipid metabolism and adipocyte differentiation. OBJECTIVE: The aim of this study was to evaluate the levels of miR-34a and miR-149 and their relationship with metabolic parameters in obese children and adolescents. DESIGN: Seventy children and adolescents were enrolled in the study. Plasma levels of microRNAs were evaluated by real-time PCR using SYBR green and analyzed by ΔCt method. Plasma concentrations of visfatin and insulin were measured by ELISA method. Glucose and lipid profile were determined colorimetrically. HOMA-IR was calculated and used as an index of insulin resistance (IR). RESULTS: miR-34a was significantly lower in subjects with insulin resistance compared to obese children with normal insulin sensitivity. There was an inverse relationship between miR-34a levels and both insulin and HOMA-IR. On the other hand, miR-149 was significantly correlated with visfatin. There was no significant difference in miR-34a and miR-149 between obese and normal weight subjects. CONCLUSIONS: miR-34a is associated with insulin and HOMA-IR and thus seems to be involved in IR. miR-149 is inversely associated with visfatin levels which could be indicative of anti-inflammatory effect of this miRNA.

The effects of Nobiletin, Hesperetin, and Letrozole in a combination on the activity and expression of aromatase in breast cancer cells.

Nobiletin (NOB) and hesperetin (HES) are the citrus polymethoxyflavone and flavonone. Aromatase or cytochrome P450 (CYP19) enzyme is a key enzyme in estrogen biosynthesis. The objective of this study was to investigate the combinational effects of HES, NOB and letrozole (LET) as aromatase inhibitors on the activity and expression of aromatase in MCF-7 cells. In this study, aromatase enzyme activity based on the conversion of androgen substrate testosterone to 17ß-Estradiol was determined. Estradiol concentrations were measured using an electrochemiluminescence immunoassay. CYP19 gene expression was determined by quantitative real-time PCR. Our findings demonstrated that none of combinations including LET+NOB, LET+HES, LET+NOB+HES, and NOB+HES had no significant effects on aromatase activity and expression. The present study showed for the first time that the combination of HES, NOB, and LET had no effects on activity and expression of aromatase in MCF-7 breast cancer cells.

Effects of Quercetin on Adiponectin-Mediated Insulin Sensitivity in Polycystic Ovary Syndrome: A Randomized Placebo-Controlled Double-Blind Clinical Trial.

Polycystic ovary syndrome (PCOS) is a heterogeneous, multi-causal, and genetically complex disorder, which is related to the failure in endocrine glands. Adiponectin has been reported to be low in PCOS, even in the absence of adiposity. Quercetin reduces serum glucose, insulin, triglycerides, and cholesterol levels and increases the expression and secretion of adiponectin. The aim of this study was to determine the effect of quercetin on the adiponectin-mediated insulin sensitivity in PCOS patients. Eighty-four women with PCOS were selected and randomly assigned to 2 groups of treatment and control. The treatment group received 1 g quercetin (two 500 mg capsules) daily for 12 weeks, and the control group received placebo. In addition to anthropometric assessments, fasting serum levels of total adiponectin, high-molecular-weight (HMW) adiponectin, glucose, insulin, testosterone, LH, and SHBG were also measured at the baseline and at the end of the trial. Quercetin could slightly increase the level of adiponectin by 5.56% as compared to placebo (adjusted p-value=0.001) and HMW adiponectin by 3.9% as compared to placebo (adjusted p-value=0.017), while it reduced the level of testosterone (0.71 ng/dl in quercetin vs. 0.77 ng/dl in placebo; p<0.001) and LH (8.42 IU/l in quercetin vs. 8.68 IU/l in placebo; p=0.009). HOMA-IR levels were also significantly (p<0.001) lower in quercetin (1.84) group compared to placebo group (2.21). Oral quercetin supplementation was effective in improving the adiponectin-mediated insulin resistance and hormonal profile of women with PCOS.

The individual or combinational effects of Hesperetin and Letrozole on the activity and expression of aromatase in MCF-7 cells.

Aromatase catalyzes the last and rate-limiting step in estrogen biosynthesis. Inhibition of estrogen production is a common strategy for breast cancer treatment. Citrus flavonoids have been confirmed to exhibit efficacious biological activities, particularly in cancer therapy. This study was carried out to investigate the effect of hesperetin on the activity and expression of aromatase and compare this property with letrozole as an aromatase inhibitor in MCF-7 breast cancer cell line. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays in this study demonstrated that hesperetin at a concentration of 200 µM decreased cell viability in a time dependent manner (P<0.05). Aromatase activity assay, based on 17ß-Estradiol (E2) production from testosterone, revealed that hesperetin had no effect. Real-time PCR results indicated that treatment with 1µM concentration of hesperetin for 48 h significantly decreased relative aromatase expression (P =0.004). Combination of letrozole and hesperetin also had no effect on aromatase. The changes in activity paralleled the expression of aromatase. Likely, the reduction in aromatase activity was delayed in time along with the reduction in expression ratio; however additional studies are needed to confirm this. In conclusion, the present study showed that hesperetin could decrease expression of aromatase at low concentrations in MCF-7 breast cancer cells.

Relationship between natural tooth shade and skin colour.

The purpose of this study was to assess the correlation of skin colour and tooth shade. One hundred and twenty six individuals aging between 18 to 25 years participated in this study. Colour of the maxillary central incisors was examined by VITA easy shade. Tooth shades were assigned to four ordinal values. Nivea Beauty Protect Foundation shade sample was used as a guide to assess facial skin colour Shin colours were also assigned to four ordinal values. Spearman test revealed that there was a significant relationship between tooth shade and skin colour Total co-relation factor was 51.6% (p <0 .01). Co-relation factors were 57% for women and 27% for men (p <0 .01). The highest tooth shade prevalence belonged to the second group and the highest skin colour prevalence was also in the second skin colour group.

The effect of Lactobacillus reuteri on bone morphogenetic protein-7 and beta transforming growth factor gene expressions in streptozotocin-induced diabetic rat's kidneys.

Diabetes mellitus is a serious health problem in the world and about 20 to 40% of the patients are being affected with diabetic nephropathy. The anti diabetic property of Lactobacillus reuteri (L. reuteri) has been reported. The study designed to investigate the effect of L. reuteri on the expression of BMP-7 and TGF-beta genes, the two basic factors involved in diabetic nephropathy. This experimental study was carried out in two months. For this goal thirty male Wistar rats with 12 weeks old and 200 +/- 50 g weight was divided into 5 groups, each consisting six rats. (1) Non diabetic, (2) Untreated diabetic, (3) Diabetic rats fed with L. reuteri, (4) Diabetic rats treated with insulin (4-5 U/kg/day), (5) Non diabetic rat fed with L. reuteri. Diabetes in the was induced single intraperitoneal (i.p.) injection of streptozotocin (50 mg kg(-1) b. wt). The L. reuteri 1 x 10(8) Colony Forming Units (CFU) were administered via oral gavages. After two months rats were anesthetized and blood samples collected. Serum nitric oxide (NO) levels were determined by a chemiluminescence method using NO analyzer and serum glucose by glucose oxidize method. The expression of BMP-7 and TGF-beta genes in the rat's kidneys were determined by real time PCR. Results showed that BMP-7 gene expression was increased in diabetic rats that fed with L. reuteri, while TGF-beta gene expressions were decreased. Histopathological study showed that administration of L. reuteri (1 x 10(8) CFU/rat/day) significantly reduced kidney fibrosis and increased meaningfully NO levels in diabetic rats fed with L. reuteri. It was concluded that L. reuteri increase BMP-7 gene expression and may prevents from renal damage by oxidative stress by increasing antioxidant capacity.

Application of albumin protein and indocyanine green chromophore for tissue soldering by using an IR diode laser: ex vivo and in vivo studies.

OBJECTIVE: We sought to examine the impact of different parameters of laser soldering on the thermophysical properties of the skin and to optimize these parameters for sealing a full-thickness incision in the rat skin under closed feedback control under in vivo conditions. BACKGROUND DATA: Laser tissue soldering based on protein as biologic glues and other compounds can provide greater bond strength and less collateral damage. Endogenous and exogenous materials such as indocyanine green (ICG) are often added to solders to enhance light absorption. METHODS: In ex vivo study, the temperature increase, number of scan (Ns), and scan velocity (Vs) were investigated. In ex vivo studies, four skin incisions were made over rat dorsa and were closed by using two different methods: (a) wound closure by suture and (b) closure by using an automated temperature-controlled system. An automated soldering system was developed based on a diode laser, IR detector, photodiode, digital thermocouple, and camera. The true temperature of heated tissue was determined by using a calibration software method. RESULTS: The results showed that at each laser irradiance (I), the tensile strength (σ) of incisions repaired in the static mode is higher than in the dynamic mode. It must also be noted that the tensile strength of the repaired skin wound was increased by increasing the irradiance in both static and dynamic modes. However, in parallel, an increase in the corresponding temperature was observed. The tensile strength was measured for sutured and laser-soldered tissue after 2 to 10 postoperative days. Histopathologic studies showed a better healing and less inflammatory reactions than with those caused by standard sutures after day 7. CONCLUSIONS: It is demonstrated that automated laser soldering technique can be practical provided the optothermal properties of tissue is carefully optimized.

Transplant of Primary Human Hepatocytes Cocultured With Bone Marrow Stromal Cells to SCID Alb-uPA Mice.

Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.

Characterization of skin tissue soldering using diode laser and indocyanine green: in vitro studies.

Laser tissue soldering based on protein as biological glues and other compounds can provide greater bond strength and less collateral damage. Endogenous and exogenous materials such as indocyanine green (ICG) are often added to solders to enhance light absorption. The purpose of this in vitro study was to examine the impact of different parameters of laser soldering on the thermo-physical properties of the skin. A mixture of albumin solder and ICG was prepared, and then the coated samples were irradiated by an 810 nm diode laser under different conditions. The temperature rise, number of scans (N(s)), and scan velocity (V(s)) were investigated in this study. The results showed that, at each laser irradiance (I), the tensile strength (sigma) of incisions repaired in static mode was higher than in dynamic mode and that the sigma increased with both increasing N(s) and increasing I. It is therefore important to consider the trade off between scan velocity and surface temperature for achieving an optimum operating condition.

Photopolymerization of dental resin as restorative material using an argon laser.

The effect of the 488-nm wavelength of argon laser at different power densities and irradiation times on the degree of conversion (DC), temperature rise, water sorption, solubility, flexural strength, flexural modulus, and microhardness of bisphenole A glycol dimethacrylate and triethylen glycol dimethacrylate with a mass ratio of 75:25 was studied. Camphorquinone and N,N'-dimethyl aminoethyl methacrylate were added to the monomer as a photo initiator system. The DC% of the resin was measured using Fourier transform infrared spectroscopy. The maximum DC% (50%), which was reached in 20 s, and temperature rise because of the reaction (13.5 degrees C) were both higher at 1075 than 700 mW/cm(2). Water sorption and solubility were measured according to ISO4049, which in our case were 23.7 and 2.20 microg/mm(3) at 1075 mW/cm(2), respectively. A flexural modulus of 1.1 GPa and microhardness of 19.6 kg/mm(2) were achieved above the power density. No significant difference was observed (i.e., p>0.05) for water sorption and flexural strength at 700 and 1075 mW/cm(2).

Effects of lifestyle and work-related physical activity on the degree of lumbar lordosis and chronic low back pain in a Middle East population.

The relationship between the degree of lumbar lordosis and chronic low back pain (LBP) has long been speculated. It is postulated that prolonged sitting and sedentary lifestyle might change the degree of lumbar lordosis and cause LBP. The purpose of this study was to determine the effects of lifestyle, exercise, work setting, work intensity, and other demographic factors such as age, height, weight, and gender on the degree of lumbar lordosis and occurrence of LBP. Eight hundred forty subjects between ages 20 and 65 years were equally categorized into four groups: normal male, normal female, males with LBP, and females with LBP. A questionnaire was used to obtain information about the subject's lifestyle, work setting, level of exercise, and work-related physical activity. A flexible ruler was used to measure lumbar lordosis in all subjects. The average degree of lumbar lordosis for all subjects was 37 degrees +/- 13 degrees. Females had greater lumbar lordosis (42 degrees +/- 15 degrees ) than males did (32 degrees +/- 10 degrees ). There was no significant difference in the degree of lumbar lordosis in subjects with different lifestyle (p = 0.97), level of physical activity (p = 0.36), work setting (p = 0.5), and with or without LBP (p = 0.28). The degree of lumbar lordosis was positively related with the number of pregnancies (p = 0.04, r = 0.25), age (p = 0.02, r = 0.1) and height (p = 0.0001, r = 0.31) and negatively related with weight (p = 0.04, r = 0.06) of the subjects. The likelihood of developing LBP was significantly higher in the subjects who had high work-related physical activity (p = 0.03) and those who exercised less often (p = 0.008). We found no significant relationship between LBP occurrence and the degree of lumbar lordosis (p = 0.68), work setting (p = 0.15), height (p = 0.08), weight (p =0.06), and age (p = 0.67) of the subjects. The degree of lumbar lordosis was not different between normal subjects and those with LBP. Lumbar lordosis was not affected by lifestyle, level of physical activity, or type of work setting. Although these factors have not been found to affect the degree of lumbar lordosis, some affected the occurrence of LBP. This finding indicates that the effect of these factors on LBP involves mechanisms other than changing the degree of lumbar lordosis.

The NF-kappa b repressing factor is involved in basal repression and interleukin (IL)-1-induced activation of IL-8 transcription by binding to a conserved NF-kappa b-flanking sequence element.

Interleukin (IL)-8, a prototypic chemokine, is rapidly induced by the pro-inflammatory cytokine IL-1 but is barely detectable in noninduced cells. Although there is clear evidence that the transcription factor NF-kappaB plays a central role in inducible IL-8 transcription, very little is known about the cis-elements and trans-acting factors involved in silencing of the IL-8 promoter. By sequence comparison with the interferon-beta promoter, we found a negative regulatory element (NRE) in the IL-8 promoter overlapping partially with the NF-kappaB response element. Here we show that an NF-kappaB-repressing factor (NRF) binds to the IL-8 promoter NF-kappaB-NRE. Reduction of cellular NRF by expressing NRF antisense RNA results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA. Mutation of the NRE site results in loss of NRF binding and increased basal IL-8 transcription. On the other hand IL-1-induced IL-8 transcription is decreased by mutating the NRE. These data provide evidence for a dual role of the NRF in IL-8 transcription. Although in the absence of stimulation it is involved in transcriptional silencing, in IL-1-induced cells it is required for full induction of the IL-8 promoter.

Interplay between repressing and activating domains defines the transcriptional activity of IRF-1.

Interferon regulatory factor-1 (IRF-1) is a transcriptional activator with weak activation capacity. By defining the transcriptional activation domain of IRF-1 we identified two activator fragments located between amino acids 185 and 256 functioning in an additive manner. Another fragment of IRF-1, which has no activator function alone, acts as a strong enhancer element of these activator sequences. This enhancer element resides between the activator domains and the C-terminus. In addition, we identified a novel type of inhibitory domain in the N-terminal 60 amino acids of IRF-1 which strongly inhibits its transcriptional activity. Because this fragment is conserved in all interferon regulatory factors, we found similar repression effects in the corresponding fragments in IRF-2, IRF-3 and interferon consensus sequence binding protein (ICSBP/IRF-8). Interestingly, the corresponding sequence in p48/IRF-9 is divergent, so that it does not show this inhibitory activity. A five-amino-acid sequence distinguishes the p48/IRF-9 N-terminus from the homologous parts in other interferon regulatory factors containing the repressing function. Replacing the diverged amino acids in IRF-1 with the corresponding sequence of p48/IRF-9 resulted in a loss of inhibitory activity within IRF-1. The opposing activities within interferon regulatory factors may contribute to balanced or tuned regulation of gene activation, depending on the promoter context.

Translation of NRF mRNA is mediated by highly efficient internal ribosome entry.

The ubiquitous transcription factor NRF (NF-kappaB repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-beta. NRF mRNA contains a long 5' untranslated region (5'UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5'UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5'UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5'UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.

NRF, a nuclear inhibitor of NF-kappaB proteins silencing interferon-beta promoter.

The family of NF-kappaB/rel transcription factors regulates a variety of promoters through specific DNA-binding sites. Consistent with the role of NF-kappaB activation as a response to pathogens, it regulates the expression of immune modulating cytokines like interferon-beta3 (IFN-3). Transcriptional regulation of IFN-beta is characterized by strict constitutive repression and virus-mediated activation. A nuclear protein, called NRF (NF-kappaB repressing factor) silences constitutively the IFN-beta3 promoter by binding to the negative regulatory element (NRE). This control is achieved by a direct protein-protein interaction between NRF and proximal bound NF-kappaB factors preventing the transcriptional activation. After viral induction NRF still binds but does not any more exert its specific inhibition. NRE-related sequences found in a number of promoters like those of HIV-1, HTLV-1 and the genes of the IL-2Ralpha and IL-8 constitute functionally related silencer elements which repress the constitutive enhancing activity of NF-kappaB/rel-binding sites from these promoters.

Constitutive silencing of IFN-beta promoter is mediated by NRF (NF-kappaB-repressing factor), a nuclear inhibitor of NF-kappaB.

Transcriptional regulation of the interferon-beta (IFN-beta) gene is characterized by strict constitutive repression and virus-specific activation. Previous studies have shown that the IFN-beta promoter is constitutively repressed by a negative regulatory element (NRE). Isolated NRE acts as a constitutive and position-independent silencer on the NF-kappaB-binding sites. Here, we describe the identification and functional characterization of the NRE-binding protein, called NRF (NF-kappaB-repressing factor), which abolishes the transcriptional activity of the bordering NF-kappaB- binding sites by a distance-independent mechanism. Deletion studies show that a minimal repression domain of NRF is sufficient to exert its inhibitory effect. In vitro, NF-kappaB proteins bind to purified NRF by a direct protein-protein interaction. We demonstrate that NRF is a ubiquitous and constitutive nuclear protein. In fibroblasts, the expression of the NRF antisense RNA releases the endogenous IFN-beta gene transcription. Our data strongly suggest that the NRF-mediated inhibition of NF-kappaB is a critical component of the IFN-beta gene silencing prior to viral infection.

The transcriptional silencer protein NRF: a repressor of NF-kappa B enhancers.

NF-kappa B/rel proteins are present in most cell types. In concert with other transcriptional factors they regulate a variety of genes which contribute to a wide spectrum of physiological activities like inflammation and apoptosis. An excellent example of this combinatorial regulation takes place in the IFN-beta promoter. In this promoter the fundamental regulatory elements are assembled within less than 100 base pairs including a NF-kappa B/rel enhancer and a negative regulatory element, called NRE. NRE is a member of a new class of transcriptional repressor sequences with a silencing capacity targeted to the NF-kappa B/rel enhancer. NRF is a novel transcriptional factor that binds to NRE. NRF belongs to a major class of transcriptional repressors that interact with specific promoter elements and repress transcription by separable repression domains. Such molecules have been termed active repressors, because they act by inhibitory protein-protein interaction and not simply by steric hindrance.