Signal transduction pathways of IL-1beta-mediated iNOS in pulmonary vascular smooth muscle cells.

Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.

Successful management of tracheotomized patients with chronic saliva aspiration by use of constant positive airway pressure.

OBJECTIVE: Management of chronic aspiration of saliva is a challenge to clinicians. The purpose of this report is to review the clinical course of 3 patients with tracheotomy who we have followed for at least 1 year and who have received constant positive airway pressure (CPAP) as a primary treatment for ongoing aspiration of saliva. METHODS: Retrospective chart review. RESULTS: We present here 3 patients with chronic congestion and persistent hypoxemia in whom a diagnosis of chronic aspiration of saliva was established by use of radionuclide salivagram. Each of these children had tracheotomy for treatment of airway obstruction. In an attempt to decrease chronic aspiration of saliva, we instituted constant positive pressure via tracheotomy. Repeat radionuclide salivagram performed on CPAP demonstrated a marked decrease in saliva aspiration. All patients experienced improvement in clinical symptoms and required only rare subsequent hospitalizations for respiratory disease. CONCLUSION: We suggest, based on this case series, that CPAP administered via a tracheotomy is an acceptable means of managing chronic salivary aspiration and that it may decrease respiratory complications in such patients.

Understanding airway disease in infants.

Large airway diseases manifest in ways distinct from those of small airway diseases. Noisy breathing that begins early in life suggests a congenital lesion of the large airways. The findings of elevated respiratory rate, in conjunction with subcostal retractions, hyperinflation to percussion, and musical wheezes, are diagnostic of small airway obstruction. Differentiating large from small airway disease is crucial, because each disease has a distinct diagnosis, and treatment of the 2 disease types can be quite different. When these principles are applied to a patient with wheezing or other signs of airway compromise, it becomes fairly easy to differentiate large from small airway disease. The treatment of patients with large airway disease can be substantially different from that of patients with small airway disease. Being able to differentiate the two is critically important. With the use of the history, physical examination, and radiographic evaluations described earlier, nearly every patient can be given an accurate diagnosis and treated appropriately.

Non-aspergillus allergic bronchopulmonary mycosis in a pediatric patient with cystic fibrosis.

This article describes a child with cystic fibrosis (CF) and allergic bronchopulmonary mycosis caused by Tricosporon beigelii. An 11-year-old boy with CF failed to respond to conventional treatment for a pulmonary exacerbation. Bronchial washings contained copious budding yeast forms, subsequently identified as T beigelii. Total serum immunoglobulin E was elevated and precipitating antibodies to T beigelii were positive. Together these findings support the diagnosis of allergic bronchopulmonary mycosis. The patient improved with antifungal therapy and systemic glucocorticoid therapy. The pathologic potential of yeast in the airways of patients with CF is unclear. The diagnosis of non-Aspergillus allergic bronchopulmonary mycosis requires a high degree of suspicion and has potentially important implications for the management of patients with CF.

Inhibition of protein geranylgeranylation causes a superinduction of nitric-oxide synthase-2 by interleukin-1beta in vascular smooth muscle cells.

Recently, we have designed farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298) that selectively block protein farnesylation and geranylgeranylation, respectively. In this study, we describe the opposing effects of these inhibitors on interleukin-1beta (IL-1beta)-stimulated induction of nitric-oxide synthase-2 (NOS-2) in rat pulmonary artery smooth muscle cells (RPASMC) and rat hepatocytes. Pretreatment of cells with GGTI-298 caused a superinduction of NOS-2 by IL-1beta. RPASMC treated with GGTI-298 (10 microM) prior to IL-1beta (10 ng/ml) expressed levels of NOS-2 protein five times higher than those exposed to IL-1beta alone. This superinduction of NOS-2 protein by pretreatment with GGTI-298 resulted in nitrite concentrations in the medium that were 5-fold higher at 10 ng/ml IL-1beta and 10-fold higher at 1 ng/ml IL-1beta. Furthermore, NOS-2 mRNA levels in RPASMC were also increased 6- and 14-fold (at 10 and 1 ng/ml IL-1beta, respectively) when the cells were pretreated with GGTI-298. In contrast, treatment of cells with the inhibitor of protein farnesylation, FTI-277 (10 microM), blocked IL-1beta-induced NOS-2 expression at mRNA and protein levels. Pretreatment with lovastatin, an inhibitor of protein prenylation, resulted in superinduction of NOS-2. This superinduction was reversed by geranylgeraniol, but not by farnesol, further confirming that inhibition of geranylgeranylation, not farnesylation, is responsible for enhanced NOS-2 expression. The results demonstrate that a farnesylated protein(s) mediates IL-1beta induction of NOS-2, whereas a geranylgeranylated protein(s) represses this induction.

Primary bronchomalacia in infants and children.

OBJECTIVE: To determine the natural history of primary bronchomalacia in infants and children. STUDY DESIGN: Retrospective chart review and follow-up telephone questionnaire of 17 patients with bronchoscopically confirmed primary bronchomalacia. RESULTS: All patients had initial symptoms within the first 6 months of life, and all patients were thought by their primary care physicians to have reactive airways disease. In no patient was the diagnosis of bronchomalacia considered before referral to our center. All patients had a harsh, monophonic wheeze loudest over the central airway and intermittently present between illnesses. All 17 patients had bronchomalacia of the left main-stem bronchus; two had mild tracheomalacia associated with the bronchomalacia; and one had bilateral bronchomalacia. One patient had associated laryngomalacia. Twenty-five percent of our patients had reactive airway disease in addition to bronchomalacia. With growth, all patients have shown a gradual improvement and a cessation of daily symptoms. All three patients older than the age of 5 years report limitation of vigorous exercise. CONCLUSIONS: Bronchomalacia should be considered in the differential diagnosis of the persistently wheezing infant and should be evaluated appropriately. More severe forms of bronchomalacia appear to predispose patients to exercise limitation later in life, which suggests that the lesion does not completely resolve with growth.

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells.

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L166-L171, 1996.-Interleukin-1 beta (IL-1 beta) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1 beta in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1 beta caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1 beta-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1 beta increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1 beta-mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation. Nuclear proteins from IL-1 beta-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-kappa B binding element present in the region between -242 and -42 bp. These data document that IL-1 beta, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-kappa B.

LLC-PK1 cell growth is repressed by WT1 inhibition of G-protein alpha i-2 protooncogene transcription.

The temporal expression of the early growth response gene (EGR-1) is one molecular mechanism for both maximal activation of the G alpha i-2 gene and accelerated growth in mitotically active predifferentiated LLC-PK1 renal cells. These events are dependent on an enhancer area in the 5'-flanking region of the G alpha i-2 gene that contains an EGR-1 motif (5'-CGCCCCCGC-3'). However, acquisition of the polarized phenotype in LLC-PK1 cells is accompanied by loss of EGR-1 expression and occupancy of the EGR-1 site by nuclear binding proteins other than EGR-1. We now demonstrate that one of these binding proteins is the Wilms' tumor suppressor (WT1). Furthermore, the temporal expression of WT1 in LLC-PK1 cells acquiring the polarized phenotype represses both G alpha i-2 gene activation and growth in these cells. These findings suggest the existence of differentiation-induced pathways in LLC-PK1 cells that alternatively abrogates EGR-1 and promotes WT1 gene expression, thereby modulating a target protooncogene G alpha i-2 that is participatory for growth and differentiation in renal cells. These studies emphasize the usefulness of the LLC-PK1 renal cell as a model to elucidate normal programs of genetic differentiation in which WT1 participates.

Expression of iNOS in cultured rat pulmonary artery smooth muscle cells is inhibited by the heat shock response.

The heat shock response is a highly conserved stress response known to alter patterns of gene expression in many cell types. We hypothesized that interleukin-1 beta (IL-1 beta)-mediated inducible nitric oxide synthase (iNOS) gene expression would be inhibited after induction of the heat shock response in cultured rat pulmonary artery smooth muscle cells (RPASMC). Exposure of RPASMC to sodium arsenite or heat led to expression of heat shock protein-70 (HSP-70) in a time- and concentration-dependent manner. Prior induction of the heat shock response inhibited IL-1 beta-mediated iNOS gene expression in a time- and dose-dependent manner. The inhibitory effects were not due to cytotoxicity, since cell viability was not affected by either sodium arsenite, heat, IL-1 beta, or their combination. Transcriptional analysis via transient transfection of the murine macrophage iNOS promoter [-1592 and -367 base pairs (bp)], upstream from the reporter gene luciferase, revealed that the heat shock response did not affect IL-1 beta-mediated promoter activation, as measured by luciferase activity. We conclude that induction of the heat shock response inhibits IL-1 beta-mediated iNOS gene expression in cultured RPASMC.

Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein alpha i-2 protooncogene transcription.

The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene G alpha i-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The G alpha i-2 gene 5'-flanking region contains a putative binding site (5'-CGCCCCCGC-3') for EGR-1 that may allow it to be a target gene for EGR-1 mitogenic signaling. We now demonstrate in LLC-PK1 renal cells the temporal expression of EGR-1 protein by immunoblotting and immunocytochemistry coincident with the maximal activation of the G alpha i-2 gene during cell growth. To determine whether G alpha i-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for G alpha i-2 (pRSV G alpha i-2) or EGR-1 (pRSV EGR-1) driven by a viral Rous sarcoma promoter enhancer to overexpress each protein. Following transfection, cell growth was examined in media containing either 10 or 0.1% fetal bovine serum. Only cells transfected with plasmids encoding G alpha i-2 and EGR-1 had growth rates greater than that of serum replete cohorts. To assess whether EGR-1 was contributing to the transcriptional activation of the G alpha i-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the G alpha i-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320% enhancement of G alpha i-2 transcription was found only in LLC-PK1 cells following their transfection with plasmids that contained both the EGR-1 binding site and overexpressed EGR-1 protein. Utilizing mobility shift assays, which compared nuclear extracts from cells before and after cell polarization, a probe containing the EGR-1 motif detected induced nuclear protein complexes during transcriptional activation of the G alpha i-2 gene. An anti-EGR-1 antibody specifically retarded the mobility of the induced nuclear complexes, indicating that the EGR-1 protein was a component of these complexes. These data provide direct evidence for a novel mitogenic signaling pathway coupling proximal signaling events that activate EGR-1 gene expression to a target protooncogene G alpha i-2 that is participatory for growth and differentiation in renal cells.

cAMP regulates G-protein alpha i-2 subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor.

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (Gs) and inhibitory pertussis toxin-sensitive G-proteins (Gi). Two Gi genes encoding the Gi isoforms G alpha i-2 and G alpha i-3 are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by G alpha i-2. The genes encoding these isoforms are similarly regulated in these cells during growth and differentiation but differ in response to steroid hormone signals (Holtzman, E.J., Kinane, T.B., West, K., Soper, B.W., Karga, H., Ausiello, D.A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, G alpha i-2 but not G alpha i-3 protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with G alpha i-2 or G alpha i-3 gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased G alpha i-2 transcription 3-fold but inhibited G alpha i-3 transcription by 50% at 12 h. In vivo footprinting of forskolin-treated cells was performed to examine the molecular basis for activation of the G alpha i-2 gene. Protected guanosines were identified in a 135-base pair (bp) area previously associated with enhancer activity of this gene in non-polarized cells. This DNA segment did not contain the classical cAMP response element 5'-TGACGTCA-3'. Utilizing the 135-bp DNA segment as a probe in mobility shift assays, which compared nuclear extracts from cells before and after forskolin treatment, an induced nuclear protein complex was identified. Following systematic reduction and mutation of this DNA segment, a "CCAAT" box motif was identified that bound the induced nuclear protein complex during forskolin-induced G alpha i-2 gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene.(ABSTRACT TRUNCATED AT 400 WORDS)