Novel non-canonical TGF-β signaling networks: Emerging roles in airway smooth muscle phenotype and function

The airway smooth muscle (ASM) plays an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). ASM cells express a wide range of receptors involved in contraction, growth, matrix protein production and the secretion of cytokines and chemokines. Transforming growth factor beta (TGF-β) is one of the major players in determining the structural and functional abnormalities of the ASM in asthma and COPD. It is increasingly evident that TGF-β functions as a master switch, controlling a network of intracellular and autocrine signaling loops that effect ASM phenotype and function. In this review, the various elements that participate in non-canonical TGF-β signaling, including MAPK, PI3K, WNT/β-catenin, and Ca²⁺, are discussed, focusing on their effect on ASM phenotype and function. In addition, new aspects of ASM biology and their possible association with non-canonical TGF-β signaling will be discussed.     

Comparison between sensitivity of autologous skin serum test and autologous plasma skin test in patients with Chronic Idiopathic Urticaria for detection of antibody against IgE or IgE receptor (FcεRIα)

Intradermal injection of autologous serum and plasma elicit a cutaneous reactivity in almost 45-60% of patients with Chronic Idiopathic Urticaria (CIU). This reactivity is associated with the presence of auto antibodies against IgE or IgE receptors. This study was carried out to compare the cutaneous reactivity of autologous serum and plasma skin tests in a series of patients with CIU for diagnosis of auto antibodies against IgE or IgE receptor. Fifty eight patients with CIU were injected intradermally with autologous serum and plasma (anticoagulated by citrate). Histamine was used as positive control and normal saline as negative control. The study group was checked by routine laboratory tests (CBC, U/A etc), allergens with skin prick tests, and serum IgE level, and auto antibodies against thyroid as well. Duration of urticaria was another factor which was assessed.There was no significant difference between positive ASST and positive APST patients for the above mentioned tests. 77.6% of the patients were Positive for APST and 65.5% were ASST positive. Duration of urticaria was longer in patients with positive ASST and APST than ASST and APST negative patients, although the difference was not statistically significant.Autologus serum skin test (ASST) and autologous plasma skin test (APST) could be used for estimation of duration and severity of urticaria and planning for the treatment.     

Passive stiffness of airway smooth muscle: The next target for improving airway distensibility and treatment for asthma?

Reduced airway distensibility due to increased airway stiffness is a characteristic of asthma. Airway stiffness is determined by the property and structural organization of the various elements of the airway wall, and is often divided into active and passive components. Active stiffness is thought to be associated with activation of muscle cells in the airway wall. This component of stiffness can be inhibited when active force produced by the muscle is abolished. Passive stiffness, on the other hand, is thought to stem from non-muscle component of the airway wall, especially the collagen/elastin fibrous network of the extracellular matrix within which the muscle cells are embedded. In this brief review, the notion that passive stiffness is exclusively extracellular in origin is challenged. Recent evidence suggests that a substantial portion of the passive stiffness of an in vitro preparation of tracheal smooth muscle is calcium sensitive and is regulated by Rho-kinase, although the underlying mechanism and the details of regulation for the development of this intracellular passive stiffness are still largely unknown. To reduce airway stiffness different lines of attack must be tailored to different components of the stiffness. The regulatable passive stiffness is distinct from the relatively permanent stiffness of the extracellular matrix and the stiffness associated with active muscle contraction. To improve airway distensibility during asthma exacerbation, a comprehensive approach to reduce overall airway stiffness should therefore include a strategy for targeting the regulatable passive stiffness.     

Does esophageal function vary at the striated and smooth muscle segments in functional chest pain?

OBJECTIVE: Hypersensitivity of the esophageal wall may contribute to the pathogenesis of functional chest pain. Whether the hypersensitivity is more uniformly distributed along the esophageal wall or is segmental is not known. METHODS: Graded balloon distentions were performed randomly at the smooth muscle as well as at the striated muscle portions of the esophagus in 20 patients with functional chest pain and in 15 healthy volunteers, using impedance planimetry. Sensory thresholds and cross-sectional area were examined in relation to the esophageal wall tension, and the results were compared between two levels as well as the two groups of subjects. RESULTS: During balloon distention, 17 (85%) patients reported typical chest pain, 11 (55%) at both levels, four (20%) at the smooth muscle level, and two (10%) at the striated muscle level only. The sensory thresholds for perception, discomfort, or pain were lower in patients than in controls (p < 0.05). The cross-sectional area and the esophageal wall stiffness at the smooth muscle level were lower than those obtained at the striated muscle level both in controls and in patients (p < 0.01). The wall tension at which moderate discomfort and pain were reported was lower in patients than controls (p < 0.05). CONCLUSIONS: Although in most patients the esophagus is uniformly hypersensitive, in some either the smooth muscle or the striated muscle segment can be hypersensitive. If considering balloon distention at only one level, we recommend balloon placement at 10 cm above the lower esophageal sphincter because of a higher yield of hypersensitivity.     

Impaired FcεRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and β-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.     

Role of sphingosine-1-phosphate inβ-adrenoceptor desensitization via Ca(2+) sensitization in airway smooth muscle

BackgroundThe correlation between inflammatory cells and airway smooth muscle plays fundamental roles in the pathophysiology of asthma. This study was designed to determine whether pre-exposure of airway smooth muscle to sphingosine-1-phosphate (S1P), which is released from mast cells by allergic reactions, causes a deterioration of β-adrenoceptor function.MethodsIsometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F₃₄₀/F₃₈₀), an indicator of intracellular Ca^2 + levels, were simultaneously measured using fura-2 loaded guinea-pig tracheal tissues. Intracellular cAMP levels were also measured.ResultsPre-exposure to S1P caused a reduction in the inhibitory effects of 0.3 μM isoprenaline, a β-adrenoceptor agonist, and 10 μM forskolin, a direct activator of adenylyl cyclase, against 1 μM methacholine-induced contraction in concentration- and time- dependent manners. In contrast, the values of F340/F380 were not augmented under this experimental condition. After incubation with S1P in the presence of 0.001-1 μM Y-27632, a Rho-kinase inhibitor, the reduced responsiveness to forskolin induced by S1P was reversed in a concentration-dependent manner. Moreover, pre-treatment with pertussis toxin (PTX), an inhibitor of Gi, suppressed the loss of forskolin-induced relaxation induced by S1P. Pre-exposure to S1P markedly inhibited the augmentation of cAMP accumulation induced by forskolin. However, addition of Y-27632 and pre-exposure to PTX returned forsokin-induced cAMP accumulation to the control level.ConclusionsPre-exposure to S1P causes heterologus desensitization of β-adrenoceptors by increasing the sensitivity of airway smooth muscle to intracellular Ca^2 +. Ca^2 + sensitization regulated by Gi and Rho-kinase is involved in this phenomenon.     

~(103)Pd radioactive stent inhibits biliary duct restenosis and reduces smooth muscle actin expression during duct healing in dogs

Introduction In recent years, the metal internal stent is widely used in benign or malignant stenosis of the esophagus, bronchus, biliary duct and blood vessels.[1, 2] Especially in the biliary stenosis caused by benign or malignant biliary disease, the internalstent not only resolves the stenosis, but also provides the conditions for further treatment, such as by radiotherapy.[3, 4] However, the 7%-42% restenosis rate limits the clinical use of the metal internal stent.[5] Treatment of biliar…     

Renin- and prorenin-induced effects in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor: does (pro)renin-(pro)renin receptor interaction actually occur?

Renin/prorenin binding to the (pro)renin receptor ([P]RR) results in direct (angiotensin-independent) second-messenger activation in vitro, whereas in vivo studies in rodents overexpressing prorenin (≈400-fold) or the (P)RR do not support such activation. To solve this discrepancy, DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release were evaluated in wild-type and human (P)RR-overexpressing vascular smooth muscle cells after their incubation with 1 to 80 nmol/L of (pro)renin. Human prorenin (4 nmol/L, ie, ≈800-fold above normal) + angiotensinogen increased DNA synthesis in human (P)RR cells only in an angiotensin II type 1 receptor-dependent manner. Prorenin at this concentration also increased plasminogen-activator inhibitor 1 release via angiotensin. Prorenin alone at 4 nmol/L was without effect, but at 20 nmol/L (≈4000-fold above normal) it activated extracellular signal-regulated kinase 1/2 directly (ie, independent of angiotensin). Renin at concentrations of 1 nmol/L (≈2000-fold above normal) and higher directly stimulated DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release in wild-type and human (P)RR cells, and similar effects were seen for rat renin, indicating that they were mediated via the rat (P)RR. In conclusion, angiotensin generation depending on prorenin-(P)RR interaction may occur in transgenic rodents overexpressing prorenin several 100-fold. Direct (pro)renin-induced effects via the (P)RR require agonist concentrations that are far above the levels in wild-type and transgenic rats. Therefore, only prorenin (and not [P]RR) overexpression will result in an angiotensin-dependent phenotype, and direct renin-(P)RR interaction is unlikely to ever occur in nonrenin-synthesizing organs.     

The significance of the nuclear farnesoid X receptor (FXR) in β cell function

Bile acids (BAs) are important signaling molecules that are involved in the regulation of their own metabolism, lipid metabolism, energy expenditure and glucose homeostasis. The nuclear farnesoid X receptor (FXR) and the G-protein-coupled TGR-5 are the most prominent BA receptors. FXR is highly expressed in liver and activation of liver FXR profoundly affects glucose homeostasis. Strikingly, the effect of FXR activation on glucose metabolism seems to depend on the nutritional status of the organism, i.e., slimness or obesity. Recently, it became evident that FXR is present in pancreatic β cells and that activation of β cell FXR contributes to the regulation of glucose homeostasis. Interestingly, FXR activation increases glucose-induced insulin secretion by non-genomic effects on stimulus-secretion coupling. The first chapter of this review shortly introduces the role of liver FXR in glucose metabolism, the second part focuses on the impact of FXR in lean and obese animals, and the third chapter highlights the significance of FXR in β cells.     

The significance of the nuclear farnesoid X receptor (FXR) in β cell function

Bile acids (BAs) are important signaling molecules that are involved in the regulation of their own metabolism, lipid metabolism, energy expenditure and glucose homeostasis. The nuclear farnesoid X receptor (FXR) and the G-protein-coupled TGR-5 are the most prominent BA receptors. FXR is highly expressed in liver and activation of liver FXR profoundly affects glucose homeostasis. Strikingly, the effect of FXR activation on glucose metabolism seems to depend on the nutritional status of the organism, i.e., slimness or obesity. Recently, it became evident that FXR is present in pancreatic β cells and that activation of β cell FXR contributes to the regulation of glucose homeostasis. Interestingly, FXR activation increases glucose-induced insulin secretion by non-genomic effects on stimulus-secretion coupling. The first chapter of this review shortly introduces the role of liver FXR in glucose metabolism, the second part focuses on the impact of FXR in lean and obese animals, and the third chapter highlights the significance of FXR in β cells.     

Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR)

Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses.     

Expression feature of CD3, FcεRIγ, and Zap-70 in patients with chronic lymphocytic leukemia

In leukemia patients, T-cell function has been suppressed with the disease progress. Patients with chronic lymphocytic leukemia (CLL) are all to a degree immunodeficient. In order to elucidate the feature of T-cell receptor signal transduction in CLL, the expression levels of CD3γ, δ, ε, and ζ chain, FcεRIγ, and Zap-70 genes in peripheral blood mononuclear cells (PBMCs) were analyzed. Real-time polymerase chain reaction with SYBR Green technique was used for detecting the gene expression level in PBMCs from 13 patients with CLL, 13 healthy individuals, and 10 B-cell acute lymphocytic leukemia (B-ALL) served as control. The β2-microglobulin gene was used as an endogenous reference. Relative mRNA expression level of genes was analyzed by using the 2(-ΔCt) × 100% method. Significant lower expression levels of CD3γ, ε, and ζ chain genes, as well as FcεRIγ gene were found in CLL samples. Moreover, there was lost the negative correlation of the expression levels between CD3ζ and FcεRIγ genes. The expression level of Zap-70 in CLL was lower than those from healthy controls, while higher than those from B-ALL group. There was no significant correlation between the expression levels of CD3ζ and Zap-70 genes neither in the healthy group nor in the CLL group. In conclusion, the results provide a global gene expression profile of CD3γ, δ, ε, and ζ chains, and the CD3ζ-related genes FcεRIγ and Zap-70 in CLL. Deficiency of these gene expression levels might represent the feature related to T-cell immunodeficiency. The study might contribute to better understand the cellular immune features in CLL patients.     

Modulation of the airway smooth muscle phenotype in a murine asthma model and effects of nuclear factor-κB inhibition

Objective: Phenotype modulation of airway smooth muscle (ASM) is a unique characteristic of asthma and is considered to regulate airway remodeling, airway hyperresponsiveness (AHR) and inflammation. The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in phenotypic modulation. Thus, models of acute and chronic asthma were established and pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor was delivered by intraperitoneal injection. Methods: The Penh value was measured using the BUXCO WBP system. Lung tissues were subjected to histologic analysis. Phenotypic markers of ASM and COL1A1 mRNA levels were measured by RT-PCR. Expression levels of phosphorylated p65 (pP65) and α-SMA were detected by Western blot. Serum cytokine levels were quantified by RayBiotech ELISA array. Results: PDTC intervention decreased the Penh values in both the acute and chronic models. The ASM area and the airway collagen area were decreased in the PDTC intervention group. A decrease in phenotypic markers were detected in both the acute and chronic models in time-dependent manner, and PDTC intervention partially reversed the phenotypic modulation. The effect of PDTC intervention on systemic inflammation was also verified. Conclusion: These results revealed the existence of a dynamic ASM phenotype modulation procedure in asthma development and that targeting NF-κB by PDTC was effective to mitigate ASM phenotype modulation and major asthmatic pathological features.     

Fcγ receptor IIB (FcγRIIB) maintains humoral tolerance in the human immune system in vivo

Maintenance of immunological tolerance is crucial to prevent development of autoimmune disease. The production of autoantibodies is a hallmark of many autoimmune diseases and studies in mouse model systems suggest that inhibitory signaling molecules may be important checkpoints of humoral tolerance. By generating humanized mice with normal and functionally impaired Fcγ receptor IIB (FcγRIIB) variants, we show that the inhibitory Fcγ-receptor is a checkpoint of humoral tolerance in the human immune system in vivo. Impaired human FcγRIIB function resulted in the generation of higher levels of serum immunoglobulins, the production of different autoantibody specificities, and a higher proportion of human plasmablasts and plasma cells in vivo. Our results suggest that the inhibitory FcγRIIB may be an important checkpoint of humoral tolerance in the human immune system.     

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.     

The Fcγ receptor IIA R131H gene polymorphism is associated with endothelial function in patients with hypercholesterolaemia

Objective

A gene polymorphism substituting arginine (R) for histidine (H) at position 131 has been described within the Fcγ receptor IIa (FcγRIIa). The R allele is associated with increased binding of CRP and enhanced activation of monocytes. FcγRIIa is also expressed on endothelial cells, and we hypothesized this polymorphism would be associated with alterations of endothelial function.

Methods

Genomic DNA was extracted and allele-specific PCR reactions were used to determine the FcγRIIa H131R polymorphism in 78 hypercholesterolaemic subjects. Using strain gauge plethysmography, forearm blood flow (FBF) responses were determined to intra-arterial infusion of acetylcholine (ACH), for endothelium-dependent vasodilatation (EDV), to nitroprusside (NP), for endothelium-independent vasodilatation (EIV), to NG-monomethyl-l-arginine (l-NMMA), for basal NO activity, and to ACH in the presence of l-NMMA, to assess the contribution of NO release to EDV.

Results

Homozygous carriers of the H allele (n = 30) had significantly better EDV than homozygous carriers of the R allele (n = 15), while heterozygotes showed an intermediate phenotype (n = 33) (e.g. % increase of FBF to ACH 48 μg/min: 527 ± 359% in H/H versus 452 ± 262% in H/R versus 332 ± 413% in R/R, p = 0.0012 by 2-way ANOVA). EIV and basal NO activity were not affected by genotype, and co-infusion of l-NMMA abolished the differences in EDV.

Conclusions

The R allele of the FcγRIIa polymorphism is associated with impaired EDV and reduced NO activity during endothelial cell stimulation. These data suggest that the functional effects of the FcγRIIa H131R gene polymorphism previously observed in vitro translate into clinically relevant alterations of endothelial function in vivo.     

Differentiation between control subjects and patients with chronic spontaneous urticaria based on the ability of anti-IgE autoantibodies (AAbs) to induce FcεRI crosslinking,as compared to anti-FcεRIα AAbs

BackgroundThe reported prevalences of IgG autoantibodies (AAbs) to FcεRIα and IgE in sera from patients with chronic spontaneous urticaria (CSU) have varied, and these AAbs are also often observed in healthy control subjects. Regarding the histamine release activity of purified IgG from patients with CSU, the number of examined patients has been small. Thus, we sought to determine the prevalence and FcεRI crosslinking ability of these AAbs in a large number of patients with CSU and non-atopic control (NC) subjects.MethodsWe compared the concentrations of anti-IgE and anti-FcεRIα AAbs and the abilities of these AAbs to cause FcεRI aggregation in patients with CSU (n = 134) and NC subjects (n = 55) using ELISA and an in vitro elicitation test, respectively.ResultsThe concentration of anti-IgE AAbs was significantly different between the NC subjects and the CSU patients (P < 0.0001, cutoff value: 0.558 μg/mL), whereas the concentration of anti-FcεRIα AAbs was not. A significant difference in the duration of illness was noted between patients with lower and those with higher concentrations of anti-IgE AAbs relative to the cutoff value. The abilities of anti-IgE AAbs, but not anti-FcεRIα AAbs, to induce FcεRI crosslinking were significantly higher in CSU patients than in NC subjects (P = 0.0106).ConclusionsIn the Japanese population of CSU patients studied, the ability of the anti-IgE AAbs to induce FcεRI crosslinking differed significantly between NC subjects and CSU patients, suggesting the involvement of anti-IgE AAbs in the pathogenesis of CSU in the Japanese population.     

Peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) improves skeletal muscle mitochondrial function and insulin sensitivity

Proteins belonging to the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) family are key regulators of cellular energy homeostasis in a number of oxidative tissues, including skeletal muscle. While the regulation and function of PGC-1α seems central to muscle fibre plasticity in endurance exercise, the role of PGC-1β in this tissue is less clear. Wright et al. (Diabetologia, DOI: ) provide evidence for a protective effect of moderately elevated PGC-1β in electroporated rat skeletal muscle against high-fat-diet-induced insulin resistance, at least in part by promoting the oxidation of long chain acyl-CoA entities and the elimination of reactive oxygen species. These data provide important insights into the biological role of PGC-1β in skeletal muscle and imply novel therapeutic avenues for improving peripheral insulin sensitivity.