HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts.

Proinflammatory cytokines and eicosanoids are central players in intestinal inflammation. IL-1, a key cytokine associated with intestinal mucosal inflammation, induces COX-2 expression in human colonic myofibroblasts (CMF) and increased prostaglandin E(2) secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1alpha-induced cyclooxygenase-2 (COX-2) expression is the result of NF-kappaB- and ERK-mediated transcription, as well as COX-2 message stabilization, which depends on p38, MAPKAPK-2 (MK-2) and human antigen R (HuR) RNA binding protein activation. Lipoxygenase (LOX)-derived hydroxyeicosatetraenoic acids (HETEs) are elevated in IBD and colonic adenomas and "cross talk" has been observed between the COX and LOX pathways. Since COX-2 expression is primarily in CMFs in colonic adenomas, we examined the impact of LOX metabolites, particularly HETEs, on IL-1alpha-induced COX-2 expression in human CMFs. Although 5(S)-, 12(R)-, and 15(S)-HETEs alone had little to no effect on COX-2 expression, they enhanced IL-1-mediated COX-2 expression 3.6 +/- 0.5-fold. Studies utilizing heterogeneous nuclear RNA amplification and 5,6-dichloro-beta-d-ribofuranosylbenzimidazole treatment were undertaken to measure COX-2 transcription and message stabilization, respectively. We found that HETEs enhanced IL-1-induced COX-2 mRNA levels in CMF as the result of increased p38, MK-2, and HuR activity, increasing message stability greater than that observed with IL-1 alone. Thus HETEs can act synergistically with IL-1alpha to induce COX-2 expression in human CMFs. HETEs may play a role in both colonic inflammation and in increasing the risk of CRC in IBD independently and via induction of COX-2-mediated prostaglandin secretion.

Epithelial cells and their neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer.

Intestinal myofibroblasts are alpha-smooth muscle actin-positive stromal cells that exist as a syncytium with fibroblasts and mural cells in the lamina propria of the gut. Through expression and secretion of cytokines, chemokines, growth factors, prostaglandins, and basal lamina/extracellular matrix molecules, as well as expression of adhesion molecules and receptors for many of the same soluble factors and matrix, myofibroblasts mediate information flow between the epithelium and the mesenchymal elements of the lamina propria. With the use of these factors and receptors, they play a fundamental role in intestinal organogenesis and in the repair of wounding or disease. Intestinal neoplasms enlist and conscript myofibroblast factors and matrix molecules to promote neoplastic growth, carcinoma invasion, and distant metastases.

Intestinal myofibroblasts and immune tolerance.

Stromal cells, such as myofibroblasts and fibroblasts, represent a significant fraction of MHC class II-positive cells in the normal human colonic lamina propria, suggesting they may play an important role in CD4(+) T cell regulation in a tolerogenic environment. The aim of this study was to examine whether human colonic myofibroblasts (CMFs) phenotypically and functionally resemble conventional antigen-presenting cells (APCs). Our results support the hypothesis that intestinal myofibroblasts are a novel, nonprofessional APC phenotype important in modulating mucosal T cell responses. Given their strategic location, we propose that intestinal myofibroblasts play a critical role in mediating tolerance to luminal antigens.

Hyposmolality stimulates Na(+)/H(+) exchange and HCO(3)(-) absorption in thick ascending limb via PI 3-kinase.

The signal transduction mechanisms that mediate osmotic regulation of Na(+)/H(+) exchange are not understood. Recently we demonstrated that hyposmolality increases HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through stimulation of the apical membrane Na(+)/H(+) exchanger NHE3. To investigate the mechanism of this stimulation, MTALs from rats were isolated and perfused in vitro with 25 mM HCO(3)(-)-containing solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (100 nM) and LY-294002 (20 microM) blocked completely the stimulation of HCO(3)(-) absorption by hyposmolality. In tissue strips dissected from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs, hyposmolality increased PI 3-K activity 2. 2-fold. Wortmannin blocked the hyposmolality-induced PI 3-K activation. Further studies examined the interaction between hyposmolality and vasopressin, which inhibits HCO(3)(-) absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO(3)(-) absorption, due to an effect of cAMP to inhibit hyposmolality- induced activation of PI 3-K. In contrast to their effects to block stimulation by hyposmolality, PI 3-K inhibitors and vasopressin have no effect on inhibition of apical Na(+)/H(+) exchange (NHE3) and HCO(3)(-) absorption by hyperosmolality. These results indicate that hyposmolality increases NHE3 activity and HCO(3)(-) absorption in the MTAL through activation of a PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP. Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated through different signal transduction mechanisms.

MAPK activation determines renal epithelial cell survival during oxidative injury.

Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.

Hypertonicity activates MAP kinases and inhibits HCO-3 absorption via distinct pathways in thick ascending limb.

Mitogen-activated protein (MAP) kinases are activated by osmotic stress in a variety of cells, but their function and regulation in renal tubules is poorly understood. The present study was designed to examine the osmotic regulation of MAP kinases in the medullary thick ascending limb (MTAL) of the rat and to determine their possible role in the hyperosmotic inhibition of HCO-3 absorption in this segment. Tissues from the inner stripe of the outer medulla and microdissected MTALs were incubated at 37 degreesC in control (290 mosmol/kgH2O) or hyperosmotic (300 mM added mannitol) solution for 15 min. Activities of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase were then measured using immune complex assays. Hyperosmolality increased p38 MAP kinase activity (2.3-fold) and ERK activity (2.0-fold) but had no effect on JNK activity (1.1-fold). Exposure to hyperosmolality for various times showed that the activation of p38 MAP kinase was rapid (