Renal pathology resulting from PGHS-2 gene ablation in DBA/B6 mice

Kidneys of prostaglandin H synthase-2 (PGHS-2) null mice fail to develop normally, leading to renal insufficiency. We have found that in a mixed DBA/B6 background, the lack of a functional PGHS-2 gene causes less severe renal pathology than was reported previously for PGHS-2 null mice in a B6 genetic background. The increase in blood urea nitrogen in the DBA/B6 strain of PGHS-2 null mice was significantly lower than reported for B6 PGHS-2 null mice (200% versus 270%). Cystic changes in DBA/B6 PGHS-2 null mice were also less severe. The DBA/B6 PGHS-2 null adult mice did not die from renal failure, unlike their B6/PGHS-2 counterparts that showed excessive neonatal and adult deaths. Therefore, DBA/B6 PGHS-2 null may be highly suitable to study the functional consequences of the lack of PGHS-2 in the kidney due to their less severe pathology and greater survival.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Transcriptional regulation of cyclooxygenase-2 in the Human Microvascular Endothelial Cell Line,HMEC-1: Control by the combinatorial actions of AP2, NF-IL-6 and CRE elements

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.     

Disease pathways at the Rat Genome Database Pathway Portal: genes in context-a network approach to understanding the molecular mechanisms of disease

Background

Biological systems are exquisitely poised to respond and adjust to challenges, including damage. However, sustained damage can overcome the ability of the system to adjust and result in a disease phenotype, its underpinnings many times elusive. Unraveling the molecular mechanisms of systems biology, of how and why it falters, is essential for delineating the details of the path(s) leading to the diseased state and for designing strategies to revert its progression. An important aspect of this process is not only to define the function of a gene but to identify the context within which gene functions act. It is within the network, or pathway context, that the function of a gene fulfills its ultimate biological role. Resolving the extent to which defective function(s) affect the proceedings of pathway(s) and how altered pathways merge into overpowering the system's defense machinery are key to understanding the molecular aspects of disease and envisioning ways to counteract it. A network-centric approach to diseases is increasingly being considered in current research. It also underlies the deployment of disease pathways at the Rat Genome Database Pathway Portal. The portal is presented with an emphasis on disease and altered pathways, associated drug pathways, pathway suites, and suite networks.

Results

The Pathway Portal at the Rat Genome Database (RGD) provides an ever-increasing collection of interactive pathway diagrams and associated annotations for metabolic, signaling, regulatory, and drug pathways, including disease and altered pathways. A disease pathway is viewed from the perspective of networks whose alterations are manifested in the affected phenotype. The Pathway Ontology (PW), built and maintained at RGD, facilitates the annotations of genes, the deployment of pathway diagrams, and provides an overall navigational tool. Pathways that revolve around a common concept and are globally connected are presented within pathway suites; a suite network combines two or more pathway suites.

Conclusions

The Pathway Portal is a rich resource that offers a range of pathway data and visualization, including disease pathways and related pathway suites. Viewing a disease pathway from the perspective of underlying altered pathways is an aid for dissecting the molecular mechanisms of disease.

     

The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.     

MOET: a web-based gene set enrichment tool at the Rat Genome Database for multiontology and multispecies analyses

Biological interpretation of a large amount of gene or protein data is complex. Ontology analysis tools are imperative in finding functional similarities through overrepresentation or enrichment of terms associated with the input gene or protein lists. However, most tools are limited by their ability to do ontology-specific and species-limited analyses. Furthermore, some enrichment tools are not updated frequently with recent information from databases, thus giving users inaccurate, outdated or uninformative data. Here, we present MOET or the Multi-Ontology Enrichment Tool (v.1 released in April 2019 and v.2 released in May 2021), an ontology analysis tool leveraging data that the Rat Genome Database (RGD) integrated from in-house expert curation and external databases including the National Center for Biotechnology Information (NCBI), Mouse Genome Informatics (MGI), The Kyoto Encyclopedia of Genes and Genomes (KEGG), The Gene Ontology Resource, UniProt-GOA, and others. Given a gene or protein list, MOET analysis identifies significantly overrepresented ontology terms using a hypergeometric test and provides nominal and Bonferroni corrected P-values and odds ratios for the overrepresented terms. The results are shown as a downloadable list of terms with and without Bonferroni correction, and a graph of the P-values and number of annotated genes for each term in the list. MOET can be accessed freely from https://rgd.mcw.edu/rgdweb/enrichment/start.html.