p38 Triggers late preconditioning elicited by anisomycin in heart: involvement of NF-kappaB and iNOS.

We investigated the role of stress-activated p38 MAP kinase (p38/SAPK-2) signaling in delayed preconditioning of the heart. Adult male out-bred ICR mice were treated with p38 activator, anisomycin (0.1 mg/kg IP), or vehicle (5% DMSO). Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to 30 minutes of ischemia and 30 minutes of reperfusion. Improvement in postischemic recovery of end-diastolic pressure and reduction in infarct size was observed, which was abolished by SB203580, a specific p38 inhibitor, and pyrrolidinediethyldithiocarbamate (PDTC), the NF-kappaB inhibitor, but not by PD 98059, a specific inhibitor for MEK1 or 2. Transient increase in p38 phosphorylation was observed 15 minutes after anisomycin treatment which subsided by 30 minutes. Electrophoretic mobility shift assay demonstrated rapid activation of NF-kappaB DNA binding with anisomycin, peaking at 30 minutes. Western blot confirmed the accumulation of p50 and p65 in nuclear extracts after anisomycin treatment. Anisomycin-induced NF-kappaB DNA binding activity was inhibited by SB203580 and PDTC. Expression of inducible nitric oxide synthase (iNOS) mRNA, protein, and nitric oxide (NO) synthesis were enhanced in anisomycin-treated mice. SB203580 and PDTC blocked the increased expression of iNOS and increase in synthesis of NO. Selective iNOS inhibitor S-methylisothiourea abolished the protective effect of anisomycin. Furthermore, postischemic cardioprotective effect of anisomycin was absent in mice with targeted ablation of iNOS gene but not in the wild-type B6.129 mice. For the first time, these results suggest that direct pharmacological activation of p38 triggers delayed preconditioning by signaling mechanism involving NF-kappaB activation and synthesis of NO from iNOS.

Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells.

Inflammatory cytokines, interleukin 1beta and tumor necrosis factor-alpha, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A(2) (PLA(2)). Cytokine-induced up-regulation of PLA(2) has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kappaB (NF-kappaB), suggesting a role for NF-kappaB in the regulation of group IIA PLA(2) expression. Reactive oxygen species such as H(2)O(2), which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-kappaB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-alpha has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA(2) up-regulation by attenuating NF-kappaB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA(2) mRNA and extracellular enzymatic activity over time. DPI (0.02-20 microM), added 90 min before cytokine activation, inhibited both group IIA PLA(2) mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-kappaB binding to one of two NF-kappaB consensus elements in the rat group IIA PLA(2) promoter and also was suppressed by DPI pretreatment. Antibodies to NF-kappaB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-kappaB species as the classical p50/p65 heterodimer.

Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures.

PURPOSE: Studies on radiation-induced changes in gene expression are likely to be very important in developing a better understanding of cellular responses to ionizing radiation. While there is some information on the activation of cellular signal transduction pathways after radiation, few late reacting target genes have been identified. This study focuses on the characterization of expression modulation of two critical growth regulatory genes, estrogen receptor and epidermal growth factor-receptor in malignant mammary epithelial cells in response to single and repeated ionizing radiation exposures. METHODS AND MATERIALS: MCF-7 cells were used for single radiation exposure (2-50 Gy) experiments and MCF-IR-3 cells, generated by exposure to cumulative doses of 60 Gy in 2 Gy fractions, respectively, were used to study the effects of repeated exposures. Steady-state messenger ribonucleic acid levels for estrogen receptor, epidermal growth factor-receptor, and transforming growth factor-alpha were determined by ribonucleic acid protection experiments. Estrogen receptor and epidermal growth factor-receptor protein expression was quantitated by competitive binding studies with 3H-estradiol and 125I-EGF. RESULTS: MCF-IR-3 cells showed a permanent three-fold down-regulation of the estrogen receptor messenger ribonucleic acid and protein, while epidermal growth factor-receptor was upregulated about nine-fold. Epidermal growth factor-receptor was substantially up-regulated in MCF-7 cells, at both the mRNA and protein levels, within 24 h of a single 2 Gy exposures, while there was a two-fold concomitant increase in transforming growth factor-alpha messenger ribonucleic acid expression. A decrease in estrogen receptor messenger ribonucleic acid and protein was suggested only after higher doses of single radiation exposures. CONCLUSION: Single and repeated radiation exposures modulate the expression of two critical growth promoting genes, estrogen receptor and epidermal growth factor-receptor, in MCF-7 cells. The inverse expression of estrogen receptor and epidermal growth factor-receptor established for estrogen receptor-positive malignant mammary epithelial cells is maintained in MCF-7 cells after single and repeated exposures suggesting that radiation acts through common regulatory circuits and may modulate the cellular phenotype.

Expression of insulin-like growth factor-1 in uremic rats: growth hormone resistance and nutritional intake.

This study was designed to test the hypothesis that induction of insulin-like growth factor-1 (IGF-1) is reduced in the uremic rat liver, which would help to explain the purported growth hormone resistance noted in uremia. IGF-1 mRNA, in the steady state and after acute induction by two doses of 100 micrograms/100 g body wt recombinant human growth hormone (rhGH), was quantitated by solution hybridization in total liver RNA, extracted by the guanidine thiocyanate/cesium chloride gradient method. Eighteen Sprague-Dawley rats weighing 100 to 102 g were randomly divided into three groups: sham-operated control rats (control group); 5/6 nephrectomized rats (uremic group); and sham-operated controls with dietary intake matching that of the uremic rats (pair-fed group). The results showed that the steady state liver IGF-1 mRNA was 1.7 arbitrary densitometry units (ADU) in the uremic animals, and was lower than the value of 3.2 ADU in the control animals (P < 0.05). After the acute administration of rhGH, the liver IGF-1 mRNA of control, uremic and pair-fed groups showed mean increases of 154% (P < 0.05), 124% (not significant, NS) and 117% (NS), respectively. The lack of IGF-1 induction in the uremic group supported the concept of growth hormone resistance in uremia. In addition, a similar lack of induction was observed in the pair-fed group, whose food intake was 65% that of the control animals. This indicated that the lack of IGF-1 induction was at least partially due to the reduced food intake.(ABSTRACT TRUNCATED AT 250 WORDS)