In gastroesophageal reflux disease, differential gene expression in the duodenum points towards enhanced chylomicron production and secretion.

BACKGROUND: Duodenal signaling affects esophageal motility and perception, both pathophysiological factors in gastroesophageal reflux disease (GERD). Duodenal gene expression abnormalities, contributing to altered esophageal sensorimotor function, have not been reported to date. AIM: To identify differentially expressed genes in GERD patients' duodenum. METHODS: Twenty GERD patients (total 24-h acid exposure 6-12%, SAP ≥95%) and ten healthy controls (HC) were included. Two weeks prior to duodenal biopsy collection, ten patients discontinued proton pump inhibitor (PPI) treatment and ten took maximum dose PPI. RNA was profiled on an Affymetrix Human Genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA). Genes exhibiting a fold change ≥ 1.4 (t test p value <1E-4) were considered differentially expressed. A subset of 21 differentially expressed genes was selected for confirmatory TaqMan low-density array RT-PCR. Mucosal apolipoprotein A-IV (apoA-IV) and cholecystokinin (CCK) concentrations were determined by ELISA and RIA, respectively. RESULTS: In GERD patients off PPI, 23 up- and 23 down-regulated genes relative to HC were found. In GERD patients on PPI, 33 and five genes were higher, respectively, lower expressed. The majority of up-regulated genes were associated with lipid absorption, particularly triglyceride resynthesis and intracellular vesicular transport, rate-limiting processes for chylomicron production and secretion. Differential expression of 11 genes was confirmed by RT-PCR. Mucosal apoA-IV and CCK concentrations (signaling proteins released upon chylomicron secretion) were similar in GERD patients and HC. CONCLUSIONS: The identified mRNA expression differences suggest that in GERD patients' duodenum, the chylomicron production and secretion potential is elevated, and may underlie a mechanism by which postprandial duodenal signaling contributes to GERD symptom generation.

Regulation of cyclooxygenase-2 expression by cyclic AMP.

Prostaglandins (PG) regulate many biological processes, among others inflammatory reactions. Cyclooxygenases-1 and -2 (COX-1 and COX-2) catalyse PG synthesis. Since this step is rate limiting, the regulation of COX expression is of critical importance to PG biology. Contrary to COX-1, which is constitutively expressed, COX-2 expression is subject to regulation. For example, COX-2 levels are increased in inflammatory reactions. Many signalling pathways can regulate COX-2 expression, not least those involving receptors for COX products themselves. Analysis of the intracellular signal transducers involved reveals a crucial role for cAMP, albeit as a modulator rather than direct inducer. Indeed, the influence of cAMP on COX-2 expression is complex and dependent on the cell type and cellular environment. This review aims to summarise various topics related to cAMP-dependent COX-2 expression. Firstly, the main aspects of COX-2 regulation are briefly considered. Secondly, the molecular basis for COX-2 gene (post)-transcriptional regulation is reviewed. Lastly, a detailed overview of the effects of cAMP-dependent signalling on COX-2 mRNA and protein expression in various human and rodent cells is provided. There is a large number of marketed, clinical and preclinical concepts promoting the elevation of intracellular cAMP levels for therapeutic purposes (e.g., beta(2)-agonists, PG receptor agonists, phosphodiesterase inhibitors). In this respect, the role of cAMP in the regulation of COX-2 expression, especially the human enzyme, is of significant clinical importance.

Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes.

Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue. They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGFbeta activity and also induces fibroblast to differentiate into alpha-smooth muscle actin (alphaSMA)-positive myofibroblasts. TGF-beta activity alone cannot completely account for alphaSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally alphaSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of alphaSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce alphaSMA expression; however, ET-1 did synergize with TGF-beta. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited alphaSMA expression in fibroblasts, costimulated with TGF-beta and ET-1. GM-CSF activated NFkappaB, thus interfering with TGF-beta signaling. Blocking TGFbeta and ET-1 largely impaired alphaSMA induction in co-cultures at day 7 and, in combination, almost completely prevented alphaSMA induction. Our results dissect the roles of TGF-beta and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.

Myofibroblast differentiation is induced in keratinocyte-fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1.

In wound healing epidermal-dermal interactions are known to regulate keratinocyte proliferation and differentiation. To find out how fibroblasts respond to epithelial stimuli, we characterized fibroblasts in monolayer co-culture with keratinocytes. On co-culture numerous extracellular matrix- and smooth muscle cell-associated gene transcripts were up-regulated in fibroblasts, suggesting a differentiation into myofibroblasts. Increased alpha-smooth muscle actin (alpha-SMA) protein expression in co-cultured fibroblasts started at approximately day 4, was serum-independent, but required endogenous transforming growth factor (TGF)-beta. In co-cultures, TGF-beta neutralizing monoclonal antibody strongly reduced alpha-SMA induction. Endogenous TGF-beta production and activation were increased at 24 and 48 hours, requiring, like alpha-SMA induction, close keratinocyte-fibroblast proximity. As myofibroblast differentiation only started after 4 days, we analyzed the presence of endogenous inhibitors at early time points. Blocking keratinocyte-derived interleukin (IL)-1 using IL-1 receptor antagonist, alpha-SMA expression in co-cultures was potentiated. Conversely, adding exogenous IL-1alpha completely suppressed endogenous alpha-SMA induction. In co-cultured fibroblasts strong nuclear factor-kappaB binding activity was observed from 2 hours, decreasing at 2 and 4 days, suggesting an early, IL-1-mediated inhibition of TGF-beta signaling in co-cultured fibroblasts. This biphasic differentiation event is regulated by the balance of endogenous TGF-beta and IL-1 activity and is reminiscent of myofibroblast differentiation at early and later stages of wound healing.

Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein.

Three mouse lines expressing Cre recombinase under the control of the human K14 promoter induced specific deletion of loxP flanked target sequences in the epidermis, in tongue, and thymic epithelium of the offspring where the Cre allele was inherited from the father. Where the mother carried the Cre allele, loxP flanked sequences were completely deleted in all tissues of the offspring, even in littermates that did not inherit the Cre allele. This maternally inherited phenotype indicates that the human K14 promoter is transcriptionally active in murine oocytes and that the enzyme remains active until after fertilization, even when the Cre allele becomes transmitted to the polar bodies during meiosis. Detection of K14 mRNA by RT-PCR in murine ovaries and immunohistochemical identification of the K14 protein in oocytes demonstrates that the human K14 promoter behaves like its murine homolog, thus identifying K14 as an authentic oocytic protein.