John F. Kuemmerle

Differential changes in brain-derived neurotrophic factor and extracellular signal-regulated kinase in rat primary afferent pathways with colitis

Abstract  Brain‐derived neurotrophic factor (BDNF) has been postulated to participate in inflammation‐induced visceral hypersensitivity by modulating the sensitivity of visceral afferents through the activation of intracellular signalling pathways such as the extracellular signal‐regulated kinase (ERK) pathway. In the current study, we assessed the expression levels of BDNF and phospho‐ERK in lumbosacral dorsal root ganglia (DRG) and spinal cord before and during tri‐nitrobenzene sulfonic acid (TNBS)‐induced colitis in rats with real‐time PCR, ELISA, western blot and immunohistochemical techniques. BDNF mRNA and protein levels were increased in L1 and S1 but not L6 DRG when compared with control (L1: two‐ to five‐fold increases, P < 0.05; S1: two‐ to three‐fold increases, P < 0.05); however, BDNF protein but not mRNA level was increased in L1 and S1 spinal cord when compared with control. In parallel, TNBS colitis significantly induced phospho‐ERK1/2 expression in L1 (four‐ to five‐fold, P < 0.05) and S1 (two‐ to three‐fold, P < 0.05) but not in L6 spinal cord levels. Immunohistochemistry results showed that the increase in phospho‐ERK1/2 expression occurred at the region of the superficial dorsal horn and grey commisure of the spinal cord. In contrast, there was no change in phospho‐ERK5 in any level of the spinal cord examined during colitis. The regional and time‐specific changes in the levels of BDNF mRNA, protein and phospho‐ERK with colitis may be a result of increased transcription of BDNF in DRG and anterograde transport of BDNF from DRG to spinal cord where it activates intracellular signalling molecules such as ERK1/2.

Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat

BackgroundIn humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization.MethodsColonic inflammation was induced by a single dose of tri-nitrobenzene sulfonic acid (TNBS) instilled intracolonically. The histology of the colon and the urinary bladder was examined by hematoxylin and eosin (H&E) stain. The protein expression of transient receptor potential (TRP) ion channel of the vanilloid type 1 (TRPV1) and brain-derived neurotrophic factor (BDNF) were examined by immunohistochemistry and/or western blot. The inter-micturition intervals and the quantity of urine voided were obtained from analysis of cystometrograms.ResultsAt 3 days post TNBS treatment, the protein level of TRPV1 was increased by 2-fold (p < 0.05) in the inflamed distal colon when examined with western blot. TRPV1 was mainly expressed in the axonal terminals in submucosal area of the distal colon, and was co-localized with the neural marker PGP9.5. In sensory neurons in the dorsal root ganglia (DRG), BDNF expression was augmented by colonic inflammation examined in the L1 DRG, and was expressed in TRPV1 positive neurons. The elevated level of BDNF in L1 DRG by colonic inflammation was blunted by prolonged pre-treatment of the animals with the neurotoxin resiniferatoxin (RTX). Colonic inflammation did not alter either the morphology of the urinary bladder or the expression level of TRPV1 in this viscus. However, colonic inflammation decreased the inter-micturition intervals and decreased the quantities of urine voided. The increased bladder activity by colonic inflammation was attenuated by prolonged intraluminal treatment with RTX or treatment with intrathecal BDNF neutralizing antibody.ConclusionAcute colonic inflammation increases bladder activity without affecting bladder morphology. Primary afferent-mediated BDNF up-regulation in the sensory neurons regulates, at least in part, the bladder activity during colonic inflammation.

Endogenous IGF-I and αvβ3 integrin ligands regulate increased smooth muscle growth in TNBS-induced colitis

Endogenous insulin-like growth factor-I (IGF-I) regulates intestinal smooth muscle growth by concomitantly stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented by the alpha(v)beta(3) integrin ligands vitronectin and fibronectin. IGF-I expression in smooth muscle is increased in both TNBS-induced colitis and Crohns disease. We hypothesized that intestinal inflammation increased vitronectin and fibronectin expression by smooth muscle and, along with IGF-I upregulation, increased intestinal muscle growth. Intestinal smooth muscle cells were examined 7 days following the induction of TNBS-induced colitis. Although alpha(v)beta(3) integrin expression was not altered by TNBS-induced colitis, vitronectin and fibronectin levels were increased by 80 +/- 10% and 90 +/- 15%, above control levels, respectively. Basal IGF-I receptor phosphorylation in inflamed muscle from TNBS-treated rats was increased by 86 +/- 8% over vehicle-treated controls. Basal ERK1/2, p70S6 kinase, and GSK-3beta phosphorylation in muscle cells of TNBS-treated rats were also increased by 140-180%. TNBS treatment increased basal muscle cell proliferation by 130 +/- 15% and decreased apoptosis by 20 +/- 2% compared with that in vehicle-treated controls. The changes in proliferation and apoptosis were reversed by an IGF-I receptor tyrosine kinase inhibitor or an alpha(v)beta(3) integrin antagonist. The results suggest that smooth muscle hyperplasia in TNBS-induced colitis partly results from the upregulation of endogenous IGF-I and ligands of alpha(v)beta(3) integrin that mediate increased smooth muscle cell proliferation and decreased apoptosis. This paper has identified one mechanism regulating smooth muscle hyperplasia, a feature of stricture formation that occurs in the chronically inflamed intestine of TNBS-induced colitis and potentially Crohns disease.

Increased activation of latent TGF-β1 by αVβ3 in human Crohn's disease and fibrosis in TNBS colitis can be prevented by cilengitide.

Background:Strictures develop in >30% of patients affected with Crohns disease. No available medication prevents stricture development in susceptible patients. In Crohns strictures, but not adjacent normal intestine, TGF-&bgr;1 increases in muscularis smooth muscle, increasing collagen I production and strictures. Muscle cells express &agr;V&bgr;3 integrin containing an Arg-Gly-Asp (RGD) binding domain. The aim was to determine whether increased TGF-&bgr;1 levels in strictures were the result of latent TGF-&bgr;1, which contains an RGD sequence, binding to and activation by &agr;V&bgr;3; and whether cilengitide, which is an RGD-containing &agr;V&bgr;3 integrin inhibitor, decreases TGF-&bgr;1 activation and development of fibrosis in chronic 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced colitis. Design:Muscle cells isolated from Crohns disease strictures and normal resection margin and from the colon of rats after 42 days of chronic TNBS-induced colitis were used to prepare RNA and protein lysates and to initiate primary cultures. The mechanisms leading to increased TGF-&bgr;1 activation, collagen I production, and fibrosis were examined in human muscle and in rats. Human cultured cells in vitro and rats in vivo were treated with cilengitide to determines it efficacy to decrease TGF-&bgr;1-activation, collagen production, and decrease the development of fibrosis. Results:Latent TGF-&bgr;1 is activated by the &agr;V&bgr;3 RGD domain in human and rat intestinal smooth muscles. Increased activation of TGF-&bgr;1 in Crohns disease and in TNBS-induced colitis causes increased collagen production, and fibrosis that could be inhibited by cilengitide. Conclusions:Cilengitide, an &agr;V&bgr;3 integrin RGD inhibitor, could be a novel treatment to diminish excess TGF-&bgr;1 activation, collagen I production, and development of fibrosis in Crohns disease.

Nonmuscle Myosin IIA Regulates Intestinal Epithelial Barrier in vivo and Plays a Protective Role During Experimental Colitis

The actin cytoskeleton is a critical regulator of intestinal mucosal barrier permeability, and the integrity of epithelial adherens junctions (AJ) and tight junctions (TJ). Non muscle myosin II (NM II) is a key cytoskeletal motor that controls actin filament architecture and dynamics. While NM II has been implicated in the regulation of epithelial junctions in vitro, little is known about its roles in the intestinal mucosa in vivo. In this study, we generated a mouse model with an intestinal epithelial-specific knockout of NM IIA heavy chain (NM IIA cKO) and examined the structure and function of normal gut barrier, and the development of experimental colitis in these animals. Unchallenged NM IIA cKO mice showed increased intestinal permeability and altered expression/localization of several AJ/TJ proteins. They did not develop spontaneous colitis, but demonstrated signs of a low-scale mucosal inflammation manifested by prolapses, lymphoid aggregates, increased cytokine expression, and neutrophil infiltration in the gut. NM IIA cKO animals were characterized by a more severe disruption of the gut barrier and exaggerated mucosal injury during experimentally-induced colitis. Our study provides the first evidence that NM IIA plays important roles in establishing normal intestinal barrier, and protection from mucosal inflammation in vivo.

Modulation of motor and sensory pathways of the peristaltic reflex by cannabinoids.

Cannabinoids have long been known to be potent inhibitors of intestinal and colonic propulsion. This effect has generally been attributed to their ability to prejunctionally inhibit release of acetylcholine from excitatory motor neurons that mediate, in part, the ascending contraction phase of the peristaltic reflex. In the present study we examined the effect of cannabinoids on the other transmitters known to participate in the peristaltic reflex using a three-compartment preparation of rat colon that allows separation of ascending contraction, descending relaxation, and the sensory components of the reflex. On addition to the orad motor compartment, anandamide decreased and AM-251, a CB-1 antagonist, increased ascending contraction and the concomitant substance P (SP) release. Similarly, on addition to the caudad motor compartment, anandamide decreased and AM-251 increased descending relaxation and the concomitant vasoactive intestinal peptide (VIP) release. On addition to the central sensory compartment, anandamide decreased and AM-251 increased both ascending contraction and SP release orad, and descending relaxation and VIP release caudad. This suggested a role for CB-1 receptors in modulation of sensory transmission that was confirmed by the demonstration that central addition of anandamide decreased and AM-251 increased release of the sensory transmitter, calcitonin gene-related peptide (CGRP). We conclude that the potent antipropulsive effect of cannabinoids is the result of inhibition of both excitatory cholinergic/tachykininergic and inhibitory VIPergic motor neurons that mediate ascending contraction and descending relaxation, respectively, as well as inhibition of the intrinsic sensory CGRP-containing neurons that initiate the peristaltic reflex underlying propulsive motility.

Hypercontractility of Intestinal Longitudinal Smooth Muscle Induced by Cytokines Is Mediated by the Nuclear Factor-κB/AMP-Activated Kinase/Myosin Light Chain Kinase Pathway

Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca2+/calmodulin-dependent kinase kinase (CaMKKβ) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKKβ with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1β or tumor necrosis factor-α, nuclear factor κB was activated as indicated by an increase in p65 phosphorylation and IκBα degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)–specific site (Ser485) that is distinct from the stimulatory CaMKKβ site (Thr172), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-κB activity with MG-132 (carbobenzoxy-l-leucyl-l-leucyl-l-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser485 and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IκBα complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor κB/PKA-dependent inhibition of AMPK and activation of MLCK.

Insulin-like growth factor-binding protein-5 stimulates growth of human intestinal muscle cells by activation of Gαi3

In human intestinal smooth muscle cells, endogenous insulin-like growth factor-I (IGF-I) regulates growth and IGF-binding protein-5 (IGFBP-5) expression. The effects of IGF-I are facilitated by IGFBP-5. We previously showed that IGFBP-5 acts independently of IGF-I in human intestinal muscle to stimulate proliferation and upregulate IGF-I production by activation of Erk1/2 and p38 MAPK. Thus a positive feedback loop exists between IGF-I and IGFBP-5, whereby both stimulate muscle growth and production of the other factor. In Crohns disease, IGF-I and IGFBP-5 expression are increased and contribute to stricture formation through this effect on muscle growth. To determine the signaling pathways coupling IGFBP-5 to MAPK activation and growth, smooth muscle cells were isolated from muscularis propria of human intestine and placed into primary culture. Erk1/2 and p38 MAPK activation and type I collagen production were measured by immunoblot. Proliferation was measured by [(3)H]thymidine incorporation. Activation of specific G proteins was measured by ELISA. AG1024, an IGF-I receptor tyrosine kinase inhibitor, was used to isolate the IGF-I-independent effects of IGFBP-5. IGFBP-5-induced phosphorylation of Erk1/2 and p38 MAPK and proliferation were abolished by pertussis toxin, implying the participation of Gi. IGFBP-5 specifically activated Gi3 but not other G proteins. Transfection of an inhibitory Galphai minigene specifically inhibited MAPK activation, proliferation, and both collagen-I and IGF-I production. Our results indicate that endogenous IGFBP-5 activates Gi3 and regulates smooth muscle growth, IGF-I production, and collagen production via the alpha-subunit of Gi3, independently of IGF-I, in normal human intestinal muscle cells.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

Loss of γ-cytoplasmic actin induces epithelial-to-myofibroblast transition (EmyT), which depends on activation of SRF and its cofactor, MRTF, formin-mediated actin polymerization, and activated Rho GTPase. This demonstrates a unique role of γ-cytoplasmic actin in regulating the epithelial phenotype and the suppression of EmyT.

Regulation of Gβγi-Dependent PLC-β3 Activity in Smooth Muscle: Inhibitory Phosphorylation of PLC-β3 by PKA and PKG and Stimulatory Phosphorylation of Gαi-GTPase-Activating Protein RGS2 by PKG

In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca2+ release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A1 receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495–689), a Gβγ-scavenging peptide, or Gαi minigene but not Gαq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca2+ release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gαi-GTPase-activating protein, RGS2, and enhanced association of Gαi3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gαi mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγi-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gαi-GTP. This allows RGS2 to accelerate Gαi-GTPase activity, enhance Gαβγi trimer formation, and inhibit Gβγi-dependent PLC-β3 activity.