Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells

Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Ctheta (PKCtheta), but not PKCepsilon and PKCdelta, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.     

Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines

Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32–34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and β-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7–8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk.     

A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.

We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.     

Release of the cytokines colony-stimulating factor-1, granulocyte-macrophage colony-stimulating factor, and IL-6 by cloned murine vascular smooth muscle cells

Vascular smooth muscle cells were cultured from the mesenteric arteries of MRL lpr/lpr, MRL +/+, CBA/J, or C3H/HeJ mice and evaluated for their ability to synthesize a range of cytokines. Vascular smooth muscle cells of MRL +/+, MRL lpr/lpr, and CBA/J origin released biologically significant amounts of CSF-1 and IL-6 and relatively low but detectable amounts of granulocyte macrophage-CSF (GM-CSF) but not IL-2, IL-3, or IL-4. Vascular smooth muscle cells of C3H/HeJ origin produced lower amounts of CSF-1 and IL-6, and GM-CSF was barely detectable. Production of these cytokines did not require the exogenous growth factors present in FCS and occurred, although at lower levels, in serum-free medium supplemented with insulin, transferrin, and albumin. Cloned lines of MRL +/+ vascular smooth muscle cells, with electron microscopic and immunochemical properties of vascular smooth muscle cells, produced CSF-1, IL-6, and GM-CSF, establishing that vascular smooth muscle cells were a direct source of CSF-1, IL-6, and GM-CSF. These observations highlight the need for experiments to directly address the question of whether vascular smooth muscle cells constitutively produce these cytokines under physiologic conditions in vivo and suggest that vascular smooth muscle cells may participate actively in inflammation by releasing cytokines that are active on lympho-hemopoietic and other cells.