FIZZ1 promotes airway remodeling through the PI3K/Akt signaling pathway in asthma

Found in inflammatory zone 1 (FIZZ1) plays a vital role in pulmonary inflammation and angiogenesis. In addition, FIZZ1 plays a role in the early stages of airway remodeling in asthma by increasing the expression of α smooth muscle actin (α-SMA) and type I collagen. However, the role of FIZZ1 in the airway remodeling of asthma remains unclear. In the present study, FIZZ1 was identified to be upregulated in ovalbumin (OVA)-induced asthmatic mice, along with phosphorylated protein kinase B (Akt). Following FIZZ1 recombinant protein co-culture in the murine lung epithelial-cell line, Akt phosphorylation was upregulated, however, following transfection with FIZZ1-small hairpin RNA, the phosphorylation levels were decreased. The variation in α-SMA and type I collagen expression levels was consistent with the Akt phosphorylation levels. Intratracheal administration of LY294002 and Akt inhibitor IV to the asthmatic mice was capable of reducing airway inflammation, downregulating the expression of α-SMA, type I collagen and fibronectin-1 and increasing the expression of E-cadherin. In conclusion, the present study demonstrated that FIZZ1 promoted airway remodeling in asthma via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Blocking the PI3K/Akt signaling pathway may attenuate the early stages of airway remodeling induced by OVA by regulating the abnormal process of epithelial-mesenchymal transition.


Introduction
Asthma is a chronic inflammatory disease of the bronchial airway characterized by chronic airway inflammation, airway hyperresponsiveness, mucus overproduction and airway remodeling (1,2). A previous study demonstrated that airway remodeling is a characteristic feature of asthma and may be observed from the early stages (3). Structural and functional changes to the epithelial cells are significant in the process of airway remodeling (3,4) and airway epithelial cells are able to promote the process through epithelial-mesenchymal transition (EMT) (5). During the process of EMT, the expression levels of type I collagen, fibronectin-1 and α-smooth muscle actin (α-SMA) have been observed to be upregulated, while the expression of the epithelial marker, E-cadherin, has been shown to be reduced (6). The abnormal process of EMT was observed as a central pathological and physiological characteristic in the early stages of airway remodeling (7).
Found in inflammatory zone 1 (FIZZ1) was first reported in 2000 by Holcomb et al in allergic pulmonary inflammation (8) and was shown to play a vital role in pulmonary inflammation and angiogenesis (9). Our previous study demonstrated that FIZZ1 was vital in airway remodeling in asthma and was capable of increasing the expression levels of α-SMA and type I collagen in the early stages of airway remodeling (10). However, the mechanism by which FIZZ1 functions in the process of airway remodeling remains unclear. In the present study, the hypothesis that FIZZ1 may activate the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway through promoting Akt phosphorylation in vitro was investigated. In addition, the effect that blocking the PI3K/Akt pathway has on decreasing inflammatory cell infiltration and alleviating airway remodeling via regulating the process of EMT was investigated.

Materials and methods
Animals. Specific-pathogen-free, female BALB/c mice (age, 8-10 weeks; weight, 20±2 g; Animal Experiment Center of Shandong University, Shandong, China) were sensitized on days 1 and 14 by intraperitoneal injection of 20 µg ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) and 4 mg Al(OH) 3 (Sigma-Aldrich) suspended in 0.2 ml saline. On days 21-23 following the initial sensitization, the mice were challenged for 30 min with an aerosol of 1% (wt/vol) OVA in saline using an ultrasonic nebulizer (PARI BOY SX, Starnberg, Germany), while saline alone was used to challenge the control group. LY294002 (7.5 mg/kg body weight; Cell Signaling Technology, Inc., Beverly, MA, USA), Akt inhibitor IV (5 mg/kg body weight; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or saline were administered intratracheally 2 h prior to each OVA aerosol challenge (Table I). All the animal experiments were approved by the Institutional Animal Care and Use Committee of Shandong University (Jinan, China).
Analysis of airway responsiveness. At 24 h after the last challenge, the mice were anesthetized by intraperitoneal injection of chloral hydrate (4 mg/kg body weight). Methacholine was administered at a concentration of 0, 4, 8, 12 or 16 g/l. Measurements of airway hyperresponsiveness were conducted using an animal pulmonary instrument (flexiVent, Hong Kong, China) 1 min after each dose with 2 min between doses. The results were expressed as the maximum resistance following each dose minus the baseline (saline alone) resistance.
Histological analysis. Lung tissues were fixed in 10% neutral formalin, paraffin-embedded, cut into 4-µm sections and stained with hematoxylin and eosin for examination of inflammatory cell infiltration.
Murine lung epithelial-12 (MLE-12) cell culture. The MLE-12 cell line was purchased from a cell bank (American Type Culture Collection, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium/F12 complete medium with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C and 5% CO 2 . Following a 48-h culture, the cells were seeded in 6-well culture plates.
FIZZ1 recombinant protein co-culture and FIZZ1 small hairpin RNA (shRNA) transfection. The MEL-12 cell line was cultured with FIZZ1 recombinant protein (1 µg/ml; Santa Cruz Biotechnology, Inc.), while the control group used phosphate-buffered saline instead. Subsequent to 24 h, the protein was extracted from the cell line for analysis of the Akt phosphorylation levels and the expression levels of α-SMA and type I collagen.
Lipofectamine 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to transfect the FIZZ1-shRNA plasmid to the cell line, according to the manufacturer's instructions. After 48 h of incubation, the cells were harvested for analysis of FIZZ1, p-Akt, α-SMA and type I collagen.
An empty plasmid was added to the control and was cultured for the same amount of time. The cells were then collected to analyze the expression of FIZZ1, p-Akt, α-SMA and type I collagen by western blot analysis.
Western blot analysis. In total, 30 µg protein extracted from the lung was separated by 10% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane and subjected to immunoblotting with anti-α-SMA, anti-type I collagen or anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.).
Statistical analysis. Data are presented as the mean ± SEM. Continuous variables were analyzed using the Student's t-test between the groups studied. P<0.05 was considered to indicate a statistically significant difference.

Results
Asthma model in mice. The murine asthma model was confirmed by detecting airway hyperresponsiveness and histopathology. As shown in Fig. 1A, the OVA-treated mice developed airway hyperresponsiveness to inhaled methacholine compared with the saline-challenged group. Furthermore, the OVA aerosol challenge induced the infiltration of inflammatory cells around the airway (Fig. 1B).

FIZZ1 and p-Akt expression in mice.
The expression of FIZZ1 in mouse airway epithelium cells was upregulated in the OVA-induced asthmatic mice compared with the saline control group ( Fig. 2A). In terms of protein levels, an enhancement of FIZZ1 expression and Akt phosphorylation was detected in the OVA group by western blot analysis (Fig. 2B).

Expression of FIZZ1 and Akt phosphorylation in MLE-12 cells following FIZZ1 shRNA transfection by western blot analysis.
To verify whether FIZZ1 was able to promote Akt phosphorylation, the MLE-12 cell line was cultured with FIZZ1 recombinant protein following FIZZ1 shRNA transfection and the phosphorylation levels of Akt were detected. Following FIZZ1 recombinant protein co-culture, Akt phosphorylation and α-SMA and type I collagen expression were upregulated (Fig. 3A). The level of FIZZ1 expression in the MLE-12 cells following FIZZ1 shRNA transfection was downregulated, as were the levels of Akt phosphorylation and α-SMA and type I collagen expression (Fig. 3B).

Effects of LY294002 and Akt inhibitor IV on OVA-induced inflammatory cell infiltration.
To determine the role of the PI3K/Akt signaling pathway in inflammatory cell infiltration, LY294002 and Akt inhibitor IV were administered intratracheally to the asthmatic mouse model. Histological analysis, as presented in Fig. 4, demonstrated that LY294002 and Akt inhibitor IV may attenuate inflammatory cell infiltration as compared with the OVA-induced asthmatic group.

LY294002 and Akt inhibitor IV may overcome airway remodeling.
To verify the effects of the PI3K/Akt pathway on OVA-induced airway remodeling, the expression levels of E-cadherin, fibronectin-1 and type I collagen in the airway epithelium cells were detected by immunohistochemistry following LY294002 and Akt inhibitor IV intratracheal administration. High expression levels of fibronectin-1 and type I collagen were identified in the epithelial cells of the airway in the asthmatic mice; LY294002 and Akt inhibitor IV reduced this expression compared with the OVA group. By contrast, the expression level of E-cadherin decreased in the OVA-induced mice, and LY294002 and Akt inhibitor IV were shown to upregulate the expression (Fig. 5). In addition, higher expression levels of α-SMA and type I collagen were identified at the mRNA and protein levels in the OVA-induced mice.

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Therefore, blocking the PI3K/Akt pathway may inhibit the production of α-SMA and type I collagen (Fig. 6).

Discussion
In the present study, FIZZ1 was demonstrated to be overexpressed in the OVA-induced asthmatic mouse model and was shown to promote Akt phosphorylation in vitro. By blocking the PI3K/Akt pathway, inflammatory cell infiltration in the OVA group was alleviated. In the OVA group, the expression levels of α-SMA, type I collagen and fibronectin-1 were upregulated in the epithelial cells, while the expression of E-cadherin was decreased, indicating that EMT is induced in the airways of asthmatic mice. LY294002 and Akt inhibitor IV can intervene with this EMT process by increasing E-cadherin expression and downregulating the expression of α-SMA, type I collagen and fibronectin-1. Thus, we hypothesize that FIZZ1 may promote airway remodeling in asthmatic mice via the PI3K/Akt signaling pathway, and that blocking the PI3K/Akt pathway may relieve airway remodeling by regulating the abnormal process of EMT in OVA-induced mouse models. Bronchial asthma is a chronic inflammatory disease of the airways. Airway remodeling, first reported by Huber in 1922 (11), is the main pathological feature of asthma and is the result of sustained inflammation and epithelial cell damage in response to airway injuries (12). The main characteristics of airway remodeling include epithelial detachment, subepithelial fibrosis, airway smooth muscle cell and goblet cell hypertrophy and hyperplasia. All the pathological changes are the major cause of the clinical symptoms and loss of lung function (3,13). Therefore, further studies of airway remodeling may provide a novel treatment strategy for asthma.
Previous studies have demonstrated that EMT promotes airway remodeling (7,14). In the present study, the expression levels of α-SMA, type I collagen and fibronectin-1 were observed to be significantly increased and E-cadherin expression was reduced compared with the saline control, indicating the occurrence of the EMT process.
Preliminary study has revealed that the expression of FIZZ1 was enhanced in the epithelium of the asthmatic rats, which stimulated the expression of α-SMA and type I collagen in fibroblasts (10). However, the mechanism by which FIZZ1 functions in this process remains unknown. In the present study, FIZZ1 was demonstrated to be capable of regulating this process via the PI3K/Akt signaling pathway.
PI3K has a wide range of biological effects in numerous types of immune cells. A previous study revealed that PI3K enzyme activity was increased in OVA-induced murine models of asthma, along with p-Akt, one of the downstream signaling molecules of PI3K (15). LY294002, a specific PI3K inhibitor, is able to significantly downregulate Akt phosphorylation and suppress inflammatory cell infiltration, mucus production and airway hyperresponsiveness in a murine asthmatic model (16). Inflammation and airway remodeling is reduced in PI3Kγ-deficient mice (17). The PI3K/Akt signaling pathway is important in asthma and can promote airway inflammation and hyperresponsiveness, upregulate T-helper 2 cytokine levels and increase mucus production (18). In the present study, following intratracheal administration of LY294002 and Akt inhibitor IV, the infiltration of inflammatory cells was relieved and the markers of EMT, α-SMA, type I collagen and fibronectin-1, expression levels were downregulated with E-cadherin expression increased when compared with the asthmatic mouse model. This result indicates that the PI3K/Akt pathway is involved in the EMT process. Thus, we hypothesize that by blocking the signaling pathway, the process of EMT may be interrupted, which in turn relieves airway remodeling in the mouse asthmatic model. The PI3K/Akt signaling pathway may be blocked by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate (19). PTEN protein expression and activity are decreased in allergen-induced asthma (20). Intratracheal administration of adenoviruses carrying PTEN cDNA can reduce the levels of interleukin-4 and -5 in bronchoalveolar lavage fluid, bronchial inflammation and airway hyperresponsiveness (21). A previous study demonstrated that PTEN inhibited the proliferation and migration of human airway smooth muscle mass cells by downregulating the activity of the Akt signaling pathway (22). Numerous studies indicate that PTEN plays a significant role in asthma. However, whether FIZZ1 is an upstream regulator of PTEN, that affects the process of EMT by regulating the PI3K/Akt pathway, remains unknown. Thus, this is the direction of research for future studies.
In summary, the results of the present study demonstrate that FIZZ1 promotes airway remodeling via the PI3K/Akt signaling pathway. In addition, blocking the PI3K/Akt signaling pathway may alleviate airway remodeling in the early stages by intervening with the EMT process. Antagonism of the PI3K/Akt signaling pathway may be a potentially useful strategy in the therapeutic intervention for asthma.