Rho-Kinase as a Drug Target for the Treatment of Airway Hyperresponsiveness in Asthma

Goto

Momata

et al. 2010

J. Smooth Muscle Res.

RhoA, a small GTPase, is one of the key proteins of smooth muscle contraction. In allergic asthma, an upregulation of RhoA in bronchial smooth muscle has been suggested. However, the mechanism of its upregulation has not yet been clarified. In the present study, the effects of interleukin-4 (IL-4), one of the T-helper 2 cytokines, on RhoA mRNA expression and promoter activity of RhoA gene were examined in cultured human bronchial smooth muscle cells (hBSMCs). The quantitative real-time RT-PCR analyses revealed that incubation of hBSMCs with IL-4 (10, 30 and 100 ng/mL, for 24 hr) caused an increase in RhoA mRNA in a concentration-dependent manner. In luciferase reporter gene assay using hBSMCs that were transfected with luciferase constructs and were then stimulated with IL-4 (100 ng/mL), an importance of the most proximal STAT6 binding region (78-70 bp upstream of the transcription initiation site) was suggested. It is thus possible that IL-4 is capable of upregulating RhoA by promoting its transcription in hBSMCs. The proximal STAT6 binding region is required for the IL-4-induced increase in promoter activity of the human RhoA gene.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Induction of RhoA gene expression by interleukin-4 in cultured human bronchial smooth muscle cells

Goto

Momata

et al. 2010

J. Smooth Muscle Res.

“…In contrast, however, a recent study raised a possibility that the activated STAT1 may have an ability to inhibit the expression of RhoA, 33) an important protein responsible for the AHR. 21,22,24,[34][35][36][37][38] Detailed studies are required to make clear the role of STAT1 in the development of AHR.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Signal Transducer and Activator of Transcription 6 (STAT6) and STAT1, but Not STAT3, Induced by Antigen Inhalation in Bronchial Smooth Muscles of Sensitized Mice

Biological & Pharmaceutical Bulletin

Todoroki

Misawa

2010

The dramatic increase in the number of asthma cases over the last decades is of great concern for public health in the world.1) The CD4 ϩ T helper 2 lymphocytes (Th2 cells) are closely associated with disease severity, suggesting an integral role of Th2 cells in the pathophysiology of allergic bronchial asthma.2-5) Th2 cells secrete various cytokines, termed Th2 cytokines, which cause several key features of allergic bronchial asthma, including airway hyperresponsiveness (AHR).2-5) Increasing evidence indicates that interleukin-13 (IL-13), one of the members of Th2 cytokine family, is a crucial mediator in the development of AHR. [6][7][8] In addition, another Th2 cytokine IL-4 is also believed to play a role in asthma.9-11) Interestingly, IL-4 shares many functional properties with IL-13, presumably because they share a common receptor composed of IL4Ra chain as one of the two hetero chains. 12)Most of the activities of IL-13 and IL-4 can be ascribed to the activation of signal transducer and activator of transcription 6 (STAT6). [13][14][15] Indeed, a critical role of the STAT6 signal transduction in the development of AHR has been suggested in STAT6-deficient mice. 16,17) Similarly, a cell-penetrating dominant-negative STAT6 peptide could inhibit AHR in a mouse model of allergic bronchial asthma.18) On the other hand, recent studies also indicated an implication of the STAT family of molecules other than STAT6, such as STAT1 19) and STAT3,20) in the development of AHR. However, there is little information whether or not antigen challenge really causes the in vivo activation of these STAT molecules in bronchial smooth muscles (BSMs). In the present study, activations of these STAT molecules were examined in BSMs of a murine model of allergic asthma, which has marked BSM hyperresponsiveness. 21,22) MATERIALS AND METHODS AnimalsMale BALB/c mice were purchased from the Charles River Japan, Inc. (Kanagawa, Japan) and housed in a pathogen-free facility. All animal experiments were approved by the Animal Care Committee of the Hoshi University (Tokyo, Japan).Antigen Sensitization and Challenge Preparation of a murine model of allergic bronchial asthma was performed as described previously. [21][22][23][24][25] In brief, BALB/c mice (8 weeks of age) were actively sensitized by intraperitoneal (i.p.) injections of 8 mg ovalbumin (OA; Seikagaku Co., Tokyo, Japan) with 2 mg Imject Alum (Pierce Biotechnology, Inc., Rockfold, IL, U.S.A.) on day 0 and day 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/ml) for 30 min on days 12, 16 and 20. A control group of mice received the same immunization procedure except for inhaling saline aerosol instead of OA challenge. The aerosol was generated with an ultrasonic nebulizer (Nihon Kohden, Tokyo, Japan) and introduced to a Plexiglas chamber box (130ϫ200 mm, 100 mm height) in which the mice were placed. Three hours after the last OA challenge, mice were sacrificed by exsanguination from the abdominal aorta under urethane (1.6 g/kg, i.p.; Sigma-Aldric...

“…More recently, Hattori et al (2007) found that STAT1-deficient mice failed to develop the hyperresponsiveness of nasal airways that is usually induced by topical antigen challenge. In contrast, however, a recent study raised the possibility that activated STAT1 may have the ability to inhibit the expression of RhoA (Wang and Koromilas, 2009), an important protein responsible for BSM hyperresponsiveness (Chiba et al, 1999;2005;2008;2009a;2009b;Gosens et al, 2006;Kume, 2008;Schaafsma et al, 2008). Further studies are required to make clear the role of STAT1 in the development of BSM hyperresponsiveness.…”

Section: S T a T 1 I N B S M C S T H E M S E L V E S M I G H T B E I mentioning

confidence: 98%

“…Similarly, a cell-penetrating dominant-negative STAT6 peptide could inhibit AHR in a mouse model of allergic bronchial asthma (McCusker et al, 2007). In cultured bronchial smooth muscle cells, IL-13 can activate STAT6 directly, resulting in an upregulation of RhoA (Chiba et al, 2009a;2009b), a crucial protein responsible for AHR (Gosens et al, 2006;Kume, 2008;Schaafsma et al, 2008). On the other hand, recent studies also indicated the implication of other members of the STAT family of molecules, such as STAT1 (Quarcoo et al, 2004) and STAT3 (Simeone-Penney et al, 2007), in the development of AHR.…”

Section: Introductionmentioning

confidence: 99%

Activation of signal transducer and activator of transcription factor 1 by interleukins-13 and -4 in cultured human bronchial smooth muscle cells

Todoroki

Misawa

2009

J. Smooth Muscle Res.

The family of signal transducer and activator of transcription (STAT) factors play a critical role in the signaling of many cytokines. In addition to the involvement of STAT6 in allergic bronchial asthma, both STAT1 and STAT3 have also been implicated. However, there is little information whether or not the T helper 2 cytokines, which cause several key features of allergic asthma, really induce the activation of STAT1 and/or STAT3 in bronchial smooth muscle (BSM) cells. In the present study, the effects of interleukin-13 (IL-13) and IL-4 on activation of these STAT molecules were examined in cultured human bronchial smooth muscle cells (hBSMCs). After a starvation period, the hBSMCs were treated with 100 ng/ml of IL-13 or IL-4. Total protein samples were prepared at intervals of 1, 3, 6, 12 and 24 hours after the cytokine treatment, and Western blot analyses for total and tyrosine-phosphorylated STATs molecules were conducted. As a result, ut was found that both IL-13 and IL-4 caused a significant increase in the levels of phosphorylated STAT1. Examination of the time-course revealed a peak of STAT1 phosphorylation at 1 hr after cytokine application. In contrast, neither IL-13 nor IL-4 induced phosphorylation of STAT3. Neither of these cytokines changed the protein expression of the STATs themselves. These findings suggest that STAT1, but not STAT3, might also be one of the crucial signal transducers in the development of BSM hyper-responsiveness, which is one of the causes of AHR in asthmatics.