Yaopeng Hu

Intestinal myofibroblast TRPC6 channel may contribute to stenotic fibrosis in Crohn's disease.

Background:Intestinal fibrosis is a frequent complication of Crohns disease (CD) and often leads to detrimental stricture formation. Myofibroblasts play active roles in mediating fibrotic changes in various tissues. We investigated whether transient receptor potential channels in intestinal myofibroblasts are involved in CD-associated intestinal fibrosis. Methods:An intestinal myofibroblast cell line (InMyoFibs) was stimulated with transforming growth factor-&bgr;1 (TGF-&bgr;1) to model excessive fibrosis. Biopsy samples from nonstenotic or stenotic intestinal regions from patients with CD were used for quantitative comparisons of transient receptor potential channel and fibrosis-associated factor expression levels. Results:TGF-&bgr;1 treatment transformed spindle-shaped InMyoFibs into filament-shaped cells with enhanced &agr;-actin stress fiber formation, transient receptor potential canonical (TRPC) 4 and TRPC6 messenger RNA and protein expression, and basal- and agonist-induced Ca2+ influxes. TGF-&bgr;1 also enhanced the formation of TRPC6/smooth muscle &agr;-actin, TRPC6/N-cadherin, and TRPC4/N-cadherin coimmunoprecipitates. Inhibition of TRPC6 in InMyoFibs by RNA interference or dominant-negative mutations suppressed TGF-&bgr;1-induced Ca2+ influxes, stress fiber formation, and smooth muscle &agr;-actin expression, but increased COL1A1, interleukin (IL)-10, and IL-11 expression, as well as Smad-2, ERK, and p38-MAPK phosphorylation. Similar increases in phosphorylation levels were observed with TRPC and calcineurin inhibitors. In stenotic areas in patients with CD, TRPC6, ACTA2 (smooth muscle &agr;-actin), CDH2 (N-cadherin), COL1A1, IL-10, and IL-11 were significantly increased. Conclusions:These results suggest that augmented Ca2+ influxes due to TRPC6 upregulation facilitate stress fiber formation and strengthen cell–cell interactions by negatively regulating the synthesis of antifibrotic factors in TGF-&bgr;1-treated myofibroblasts. Similar changes observed in stenotic areas of patients with CD suggest the therapeutic significance of targeting TRPC6.

Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

•  Ca2+/calmodulin (CaM)‐dependent kinase II (CaMKII) plays pivotal roles in diverse Ca2+‐mediated cellular functions including the physiology/pathophysiology of the cardiovascular system, through modulation of a variety of Ca2+‐permeable channels such as a non‐voltage‐gated Ca2+ channel TRPC6. •  In this study, we investigated the molecular mechanism underlying its positive regulation by CaMKII with chimera, deletion and site‐directed mutagenesis approaches. •  The results indicate that two spatially separated sites of TRPC6 channel, i.e. a distal part of the C‐terminal inositol‐1,4,5‐trisphosphate receptor/CaM binding domain and Thr487 located on the putative first intracellular loop, are crucial for the CaMKII‐mediated regulation of TRPC6 channels. •  This mechanism may serve as an effective positive feedback regulation of Ca2+ influx through TRPC6 channels, in concert with intracellular and transmembrane Ca2+ mobilization upon phospholipase C‐coupled receptor stimulation by neurohormonal factors, thereby fine‐tuning the cardiovascular functions. •  Disruption of these could lead to pathological states such as cardiac hypertrophy and arrhythmia, hypertension and atherosclerosis.

Significant contribution of TRPC6 channel-mediated Ca(2+) influx to the pathogenesis of Crohn's disease fibrotic stenosis.

Intestinal fibrosis is an intractable complication of Crohns disease (CD), and, when occurring excessively, causes severe intestinal obstruction that often necessitates surgical resection. The fibrosis is characterized by an imbalance in the turnover of extracellular matrix (ECM) components, where intestinal fibroblasts/myofibroblasts play active roles in ECM production, fibrogenesis and tissue remodeling, which eventually leads to the formation of stenotic lesions. There is however a great paucity of knowledge about how intestinal fibrosis initiates and progresses, which hampers the development of effective pharmacotherapies against CD. Recently, we explored the potential implications of transient receptor potential (TRP) channels in the pathogenesis of intestinal fibrosis, since they are known to act as cellular stress sensors/transducers affecting intracellular Ca2+ homeostasis/dynamics, and are involved in a broad spectrum of cell pathophysiology including inflammation and tissue remodeling. In this review, we will place a particular emphasis on the intestinal fibroblast/myofibroblast TRPC6 channel to discuss its modulatory effects on fibrotic responses and therapeutic potential for anti-fibrotic treatment against CD-related stenosis.

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches

Aims Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4. Methods and results Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent Kd = ∼500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+- and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (≥ six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced. Conclusions These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds

Transient receptor potential canonical (TRPC) proteins form Ca2+-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca2+ influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca2+ signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca2+-dependent transcription factor, cAMP response element–binding protein. These findings suggest that Ca2+ signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease

Background & Aims The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling. Methods An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn’s disease (CD) patients were used for pathologic staining and quantitative analyses. Results In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca2+ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1–induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice. Conclusions TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders.

Daikenchuto (Da-Jian-Zhong-Tang) ameliorates intestinal fibrosis by activating myofibroblast transient receptor potential ankyrin 1 channel

AIM To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto (DKT) associated with transient receptor potential ankyrin 1 (TRPA1) channels in intestinal myofibroblasts. METHODS Inflammatory and fibrotic changes were detected in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) chronic colitis model of wild-type and TRPA1-knockout (TRPA1-KO) mice via pathological staining and immunoblotting analysis. Ca2+ imaging experiments examined the effects of DKT and its components/ingredients on intestinal myofibroblast (InMyoFib) cell TRPA1 channel function. Pro-fibrotic factors and transforming growth factor (TGF)-β1-associated signaling were tested in an InMyoFib cell line by qPCR and immunoblotting experiments. Samples from non-stenotic and stenotic regions of the intestines of patients with Crohn’s disease (CD) were used for pathological analysis. RESULTS Chronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice. A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice. The active ingredients of DKT, i.e., hydroxy α-sanshool and 6-shogaol, induced Ca2+ influxes in InMyoFib, and this was antagonized by co-treatment with a selective TRPA1 channel blocker, HC-030031. DKT counteracted TGF-β1-induced expression of Type I collagen and α-smooth muscle actin (α-SMA), which were accompanied by a reduction in the phosphorylation of Smad-2 and p38-mitogen-activated protein kinase (p38-MAPK) and the expression of myocardin. Importantly, 24-h incubation with a DKT active component Japanese Pepper increased the mRNA and protein expression levels of TRPA1 in InMyoFibs, which in turn negatively regulated collagen synthesis. In the stenotic regions of the intestines of CD patients, TRPA1 expression was significantly enhanced. CONCLUSION The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis, and benefit inflammatory bowel disease treatment.

TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RT‐PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (Ispont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. Ispont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

Lipid-Mediated Mechanisms Involved in the Mechanical Activation of TRPC6 and TRPV4 Channels in the Vascular Tone Regulation

The transient receptor potential (TRP) proteins form a large Ca2+-permeable nonselective cation channel superfamily activated by physicochemical stimuli, and participate in a wide array of biological functions including sensory signal transduction. Recent investigations have disclosed that many of TRP channels expressed in the cardiovascular system (CVS) are activated by mechanical stresses operating therein such as membrane stretch, hypoosmolarity and shear stress. Although mechanisms proposed for mechanical signal transduction are diverse, accumulating evidence suggests that lipid mediators derived from phospholipase C (PLC)- and phospholipase A2 (PLA2)-dependent pathways may play central roles in the activation and regulation of these TRP channels. In this review, we focus on the lipid-mediated regulation of two TRP channels abundantly expressed in the CVS, i.e. TRPC6 and TRPV4, with particular interest in the synergistic interaction between receptor-mediated and mechanical stimulations, and discuss about their complex functional antagonism in vascular tone and blood pressure regulation.