Gene network analysis for identification of microRNA biomarkers for asthma.

BACKGROUND: To date, reliable biomarkers for asthma have not been identified. MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate post-transcriptional gene expression, and they are involved in various diseases, including asthma. MiRNAs may serve as ideal biomarkers due to their ability to regulate multiple pathways. This study aims to identify miRNA biomarker signatures for asthma. METHODS: We used the house dust mite (HDM) mouse model of allergic inflammation. Mice were phenotyped by assessing lung function, allergic response, airway inflammation, and remodeling. The miRNA signature profiles in serum and lung tissue were determined by small RNA sequencing, and data were analyzed using Qiagen CLC Genomics Workbench. To identify relevant gene targets, we performed mRNA sequencing, followed by miRNA-targets analysis. These miRNAs and targets were subject to subsequent pathway and functional analyses. RESULTS: Mice exposed to HDM developed phenotypic features of allergic asthma. miRNA sequencing analysis showed that 213 miRNAs were substantially dysregulated (FDR p-value < 0.05 and fold change expression > + 1.5 and < - 1.5) in the lung of HDM mice relative to the control mice. In contrast, only one miRNA (miR-146b-5p) was significantly increased in serum. Target analysis of lung dysregulated miRNAs revealed a total of 131 miRNAs targeting 211 mRNAs. Pathway analysis showed T helper 2/1 (Th2/Th1) as the top significantly activated signaling pathway associated with the dysregulated miRNAs. The top enriched diseases were inflammatory response and disease, which included asthma. Asthma network analysis indicated that 113 of 131 miRNAs were directly associated with asthma pathogenesis. CONCLUSIONS: These findings suggest that most dysregulated miRNAs in the HDM model were associated with asthma pathogenesis via Th2 signaling. We identified a panel of 30 miRNAs as potential biomarker candidates for asthma.

Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A.

This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Regulation of IL-17A and implications for TGF-ß1 comodulation of airway smooth muscle remodeling in severe asthma.

Severe asthma develops as a result of heightened, persistent symptoms that generally coincide with pronounced neutrophilic airway inflammation. In individuals with severe asthma, symptoms are poorly controlled by high-dose inhaled glucocorticoids and often lead to elevated morbidity and mortality rates that underscore the necessity for novel drug target identification that overcomes limitations in disease management. Many incidences of severe asthma are mechanistically associated with T helper 17 (TH17) cell-derived cytokines and immune factors that mediate neutrophilic influx to the airways. TH17-secreted interleukin-17A (IL-17A) is an independent risk factor for severe asthma that impacts airway smooth muscle (ASM) remodeling. TH17-derived cytokines and diverse immune mediators further interact with structural cells of the airway to induce pathophysiological processes that impact ASM functionality. Transforming growth factor-ß1 (TGF-ß1) is a pivotal mediator involved in airway remodeling that correlates with enhanced TH17 activity in individuals with severe asthma and is essential to TH17 differentiation and IL-17A production. IL-17A can also reciprocally enhance activation of TGF-ß1 signaling pathways, whereas combined TH1/TH17 or TH2/TH17 immune responses may additively impact asthma severity. This review seeks to provide a comprehensive summary of cytokine-driven T cell fate determination and TH17-mediated airway inflammation. It will further review the evidence demonstrating the extent to which IL-17A interacts with various immune factors, specifically TGF-ß1, to contribute to ASM remodeling and altered function in TH17-driven endotypes of severe asthma.

Differential regulation of cytokine and chemokine expression by MK2 and MK3 in airway smooth muscle cells.

BACKGROUND: Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. METHODS: Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20 h with 10 ng/ml each interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. RESULTS: Expression of these MKs and stimulation with 10 ng/ml IL-1ß, TNFα and IFNγ for 20 h did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2. CONCLUSIONS: These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 µM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 µM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Epigenetics and miRNA emerge as key regulators of smooth muscle cell phenotype and function.

Regulation of phenotypic plasticity in smooth muscle requires an understanding of the mechanisms regulating phenotype-specific genes and the processes dysregulated during pathogenesis. Decades of study in airway smooth muscle has provided extensive knowledge of the gene expression patterns and signaling pathways necessary to maintain and alter smooth muscle cell phenotype. With this solid foundation, the importance and complexity of inheritable epigenetic modifications and mechanisms silencing gene expression have now emerged as fundamental components regulating aspects of inflammation, proliferation and remodeling.

TRPC1 and Orai1 interact with STIM1 and mediate capacitative Ca(2+) entry caused by acute hypoxia in mouse pulmonary arterial smooth muscle cells.

Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca(2+) entry (CCE) but the molecular candidate(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if transient receptor potential canonical 1 (TRPC1) and Orai1 interact with stromal interacting molecule 1 (STIM1) and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. Acute hypoxia also increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+), and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive rise in [Ca(2+)](i) and the increase in Mn(2+) quench rate were both inhibited in cells treated with TRPC1 antibody or TRPC1 small interfering (si)RNA, in STIM1 siRNA-transfected cells and in Orai1 siRNA-transfected cells. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca(2+)](i) and Mn(2+) quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and in cells transfected with Orai1 siRNA. Furthermore, TRPC1 and Orai1 coimmunoprecipitated with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed colocalizations of TRPC1-STIM1 and Orai1-STIM1, and the colocalizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in acute hypoxic mouse PASMCs.

Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 µM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 µM, with or without 10 µM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 µM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease.

Variants of stretch-activated two-pore potassium channel TREK-1 associated with preterm labor in humans.

Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.

Transgenic overexpression of α7 integrin in smooth muscle attenuates allergen-induced airway inflammation in a murine model of asthma.

Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7ß1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle-specific α7 integrin transgenic mice (TgSM-Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over-expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP-1/CCL2, and MIP-1ß/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM-Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma.

T-bet is induced by interferon-γ to mediate chemokine secretion and migration in human airway smooth muscle cells.

An inappropriate balance between T-helper (Th)1 and Th2 cytokine production underlies inflammatory changes that result in airway disease. Expression of the T-box transcription factor T-bet regulates differentiation of Th cells and production of Th1 cytokines, particularly IFNγ. T-bet-deficient mice develop airway hyperreactivity, undergo airway remodeling, and exhibit defects in IFNγ production while overproducing Th2 cytokines. T-bet is also reduced in the airways of asthmatic patients, suggesting loss of T-bet expression or activity promotes development of inflammatory airway disease. We present novel data demonstrating T-bet expression is induced in human airway smooth muscle cells (ASMC) by IFNγ. This IFNγ-stimulated expression of T-bet is dependent on signaling through JAK2 and signal transducers and activators of transcription 1 (STAT1) and activates T-bet-dependent DNA binding activity. Expression of T-bet stimulates IFNγ-stimulated IFNγ expression, secretion, and promoter activity, while inhibiting IFNγ-stimulated release of chemokines including monocyte chemoattractant protein (MCP)-1/CCL2, regulated on activation normal T-expressed and secreted (RANTES)/CCL5, and eotaxin/CCL11. This is accompanied by changes in expression of the chemokine receptors CCR3 and IL12Rß2 and TNFα. T-bet expression also reduces chemotactic migration of ASMC in response to serum and PDGF, which contributes to airway hyperplasia. These results are the first to identify T-bet expression and activity in a structural cell of the lung and may provide new insights into therapeutic targets for inflammatory airway disease.

Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK.

The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1ß, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 µM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1ß, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1ß, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation.

Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice.

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac-specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxycycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure.

MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype.

Defining mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory in response to mechanical and environmental stress is crucial for determining the contribution of airway smooth muscle (ASM) to inflammatory responses that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype, but little is known about the expression and functions of miRNA in smooth muscle. In the present study we used microarrays to determine whether miRNAs in human ASM cells are altered by a proinflammatory stimulus. In ASM cells exposed to IL-1beta, TNF-alpha, and IFN-gamma, we found 11 miRNAs to be significantly down-regulated. We verified decreased expression of miR-25, miR-140*, mir-188, and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin, and TNF-alpha; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle-specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.

Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca(2+) entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

Expression profile and protein translation of TMEM16A in murine smooth muscle.

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-µm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.

Myotendinous junction defects and reduced force transmission in mice that lack alpha7 integrin and utrophin.

The alpha7beta1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of alpha7beta1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and alpha7 integrin (mdx/alpha7(-/-)), or dystrophin and utrophin (mdx/utr(-/-)), exhibit severe muscle pathology and die prematurely. To explore the contribution of the alpha7beta1 integrin and utrophin to muscle integrity and function, we generated mice lacking both alpha7 integrin and utrophin. Surprisingly, mice that lack both alpha7 integrin and utrophin (alpha7/utr(-/-)) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to alpha7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in alpha7/utr(-/-) skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The alpha7/utr(-/-) mouse, therefore, demonstrates the critical roles of alpha7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the alpha7beta1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that the TRPC1 channel mediates capacitative Ca(2+) entry (CCE), but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediates CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. In addition, CPA increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+) and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive sustained rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both inhibited in cells pretreated with antibody raised against an extracellular epitope of TRPC1. Moreover, STIM1 siRNA reduced the rise in [Ca(2+)](i) and Mn(2+) quench of fura-2 fluorescence caused by CPA, whereas overexpression of STIM1 resulted in a marked increase in these responses. RT-PCR revealed TRPC1 and STIM1 mRNAs, and Western blot analysis identified TRPC1 and STIM1 proteins in mouse PASMCs. Furthermore, TRPC1 was found to co-immunoprecipitate with STIM1, and the precipitation level of TRPC1 was increased in cells subjected to store depletion. Taken together, store depletion causes activation of voltage-operated Ca(2+) entry and CCE. These data provide direct evidence that CCE is mediated by TRPC1 channel through activation of STIM1 in mouse PASMCs.

Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling.

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice.

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.