Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa.

BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.

Short-chain fatty acids modulate gene expression for vascular endothelial cell adhesion molecules.

OBJECTIVE: Leukocyte infiltration into the intestinal wall is central to the pathogenesis of tissue injury that occurs in patients with a variety of inflammatory bowel diseases. Migration of leukocytes from the intestinal circulation into bowel tissues is mediated by chemotactic substances and adhesion molecules (i.e., intercellular adhesion molecule-1 [ICAM-1] and E-selectin) on the surface of endothelial cells lining blood vessels. Short-chain fatty acids (SCFAs) derived from dietary fiber decrease inflammatory responses in colon cells. However, the effect of SCFAs on vascular adhesion molecules is unknown. We investigated the effects of SCFAs on vascular endothelial cell adhesion molecule expression. METHODS: We assessed the effect of physiologically relevant concentrations of butyrate on expression of ICAM-1 protein and mRNA in cultures of human umbilical vein endothelial cells. We also assessed the effect of butyrate on levels of HLA-DR, E-selectin, vascular cell adhesion molecule-1, and endoglin. In additional experiments, we evaluated the effect of butyrate on ICAM-1 mRNA stability and the effect of valerate, isobutyrate, and propionate on ICAM-1 expression. The effect of butyrate on ICAM-1 expression was compared with that of trichostatin A, a specific inhibitor of histone deacetylase. Data were evaluated with Student's t tests or Tukey's multiple comparison tests, with P < 0.05 considered statistically significant. RESULTS: Butyrate concentrations of 2.5 to 5 mM significantly increased endothelial expressions of ICAM-1 protein and mRNA. The effect of butyrate (5 mM) on ICAM-1 expression was time dependent, with significant increases in levels occurring after 16 h of incubation. Butyrate (5 mM) also increased expression of E-selectin but not of HLA-DR, vascular cell adhesion molecule-1, or endoglin. Isobutyrate had little effect on ICAM-1 expression, whereas valerate and propionate significantly increased expression of ICAM-1 but were weaker stimulants compared with butyrate. Butyrate (5 mM) did not alter stability of ICAM-1 mRNA. The effect of butyrate (5 mM) was comparable to that of trichostatin A. The stimulatory effect of butyrate on ICAM-1 expression was reversed after 48 h of butyrate withdrawal. CONCLUSIONS: Butyrate increases vascular endothelial expressions of ICAM-1 and E-selectin. We speculate that butyrate-induced effects on vascular adhesion molecules modulate gut inflammation. The role of SCFAs and fiber in the pathogenesis and modulation of gut inflammation in vivo requires further study.

Identification of Barx2b, a serum response factor-associated homeodomain protein.

CC(A/T)(6)GG or serum response elements represent a common regulatory motif important for regulating the expression of many smooth muscle-specific genes. They are multifunctional elements that bind serum response factor (SRF) and are important for serum induction of genes, expression of muscle-specific genes, and differentiation of vascular smooth muscle cells. In the current study, a yeast two-hybrid screen was used to identify proteins from mouse intestine that interact with SRF. A novel homeodomain-containing transcription factor, called Barx2b, was identified that specifically interacts with SRF and promotes the DNA binding activity of SRF. Northern blotting, RNase protection analysis, and Western blotting revealed that Barx2b mRNA and protein are expressed in several smooth muscle-containing tissues, as well as in skeletal muscle and brain. In vitro binding studies using bacterial fusion proteins revealed that the DNA-binding domain of SRF interacts with a region of Barx2b located amino-terminal of the homeobox domain. The results of these studies support the hypothesis that interaction of SRF with different homeodomain-containing proteins may play a critical role in determining the cell-specific functions of SRF.

Telokin expression is restricted to smooth muscle tissues during mouse development.

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.

Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes.

Results show that smooth muscle-specific promoters represent novel downstream targets of the winged helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1). HFH-1 strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells. HFH-1 was also found to repress transcription of several other smooth muscle-specific promoters, including the SM22alpha promoter. HFH-1 inhibits telokin promoter activity, by binding to a forkhead consensus site located within an AT-rich region of the telokin promoter. The DNA-binding domain alone was sufficient to mediate inhibition, suggesting that binding of HFH-1 blocks the binding of other positive-acting factors. HFH-1 does not disrupt serum response factor binding to an adjacent CArG box within the telokin promoter, implying that HFH-1 must compete with other unidentified trans-activators to mediate repression. The localization of HFH-1 mRNA to the epithelial cell layer of mouse bladder and stomach implicates HFH-1 in repressing telokin expression in epithelial cells. This suggests that cell-specific expression of telokin is likely mediated by both positive-acting factors in smooth muscle cells and negative-acting factors in nonmuscle cell types. We propose a model in which the smooth muscle specificity of the telokin promoter is regulated by interactions between positive- and negative-acting members of the hepatocyte nuclear factor-3/forkhead family of transcription factors.

Targeted expression of SV40 large T-antigen to visceral smooth muscle induces proliferation of contractile smooth muscle cells and results in megacolon.

Many pathological conditions result from the proliferation and de-differentiation of smooth muscle cells leading to impaired contractility of the muscle. Here we show that targeted expression of SV40 large T-antigen to visceral smooth muscle cells in vivo results in increased smooth muscle cell proliferation without de-differentiation or decreased contractility. These data suggest that the de-differentiation and proliferation of smooth muscle cells, seen in many pathological states, may be independently regulated. In the T-antigen transgenic mice the increased smooth muscle cell proliferation results in thickening of the distal colon. Consequently the distal colon becomes hyper-contractile and impedes the flow of digesta through the colon resulting in enlargement of the colon oral to the obstruction. These transgenic mice thus represent a novel model of megacolon that results from increased smooth muscle cell proliferation rather than altered neuronal innervation.

Heparan-dependent endothelial antithrombin binding is increased by butyrate.

Heparin biosynthesis involves a critical early step of N-deacetylation which is inhibited by the short chain fatty acid n-butyrate. Such inhibition causes mast cells to produce heparins with high affinity for antithrombin (AT). We have cultured endothelial cells in media supplemented with short chain fatty acids and have found that isobutyric, propionic and valeric acids cause significant increases in endothelial binding of AT measured by flow cytometry, but n-butyric acid was the most effective fatty acid to increase AT binding. Such binding,was heparan sulfate-dependent, for it was decreased significantly by pre-treatment of the cells with heparinase. These findings suggest that inhibition of N-deacetylation in heparan biosynthesis affects sulfation and results in the distribution of negative charges and conformation changes within the heparan domain that binds AT to endothelial plasma membranes. These changes also were associated with up-regulation of the intercellular adhesion molecule-1, which is a marker of endothelial activation.

Monoclonal antibody anti-type I and anti-zebrin II labelling in siluriform fishes: the role of shared lineage versus shared function in polypeptide co-distributions.

Two monoclonal antibodies (mabs), the newly generated mab anti-type I and the previously described mab anti-zebrin II, were reacted with brainstem sections of two ostariophysan siluriforms, the gymnotoid Rhamphichthys rostratus and the siluroid Ictalurus punctatus. Mab anti-type I recognizes a 47 kDa polypeptide present in the dendrites and soma of projection neurons. Mab anti-zebrin II recognizes a 36 kDa polypeptide present throughout the neuronal cytoplasm, including the axon. Strongly type I immunopositive cells include: all cerebellar Purkinje cells; pyramidal cells of the nucleus medialis, electrosensory lateral line lobe and tectum; pacemaker relay cells; Mauthner neurons; lateral line ganglion cells; cells of the inferior olive; and large neurons of the reticular formation and lateral reticular nucleus. Weakly reactive type I cells include: neurons in the torus semicircularis, medial and efferent octavolateralis nuclei, the magnocellular and lateral tegmental nuclei; and the motor neurons of the Vth, VIIth and Xth cranial nerves. Most type I positive cells are brainstem projection neurons. Zebrin II expression is restricted to subsets of two cell types which also express the type I antigen - Purkinje cells and acousticolateralis pyramidal cells. Both of these neuronal types develop from the region of the rhombic lip. While the mutual expression of the type I antigen can be explained by the shared function of projection neurons, the common expression of the zebrin II antigen is most likely due to a shared embryological and/or phylogenetic lineage.