Mislocalization of Runt-related transcription factor 3 results in airway inflammation and airway hyper-responsiveness in a murine asthma model

Abstract

The Runt-related transcription factor (RUNX) gene family consists of three members, RUNX1, -2 and -3, which heterodimerize with a common protein, core-binding factor β, and contain the highly conserved Runt-homology domain. RUNX1 and -2 have essential roles in hematopoiesis and osteogenesis. Runx3 protein regulates cell lineage decisions in neurogenesis and thymopoiesis. The aim of the present study was to determine the expression features of the Runx3 protein in a murine asthma model. In vivo, Runx3 protein and mRNA were found to be almost equivalently expressed in the murine lung tissue of the control, ovalbumin (OVA) and genistein groups; however, the nuclear Runx3 protein was abated in lung tissue in OVA-immunized and challenged mice. Following treatment with genistein, which is a flavonoid previously demonstrated to decrease airway inflammation in asthma, the allergic airway inflammation and airway hyper-responsiveness were attenuated and the Runx3 protein tended to augment in the nucleus. These results were further determined in vitro. These results indicated that the mislocalization of Runx3 protein is a molecular mechanism of allergic inflammation and airway hyper-responsiveness in a murine asthma model.

Keywords: airway inflammation; asthma; genistein; interleukin-2-inducible T cell kinase inhibitor; runx3.

Figure 1.

Relief of allergic airway inflammation in mice treated with genistein. Hematoxylin and eosin staining of lung sections obtained from mice immunized and exposed to OVA and mice administered with genistein or saline. (A) Normal lungs from mice of the control group demonstrating airways and blood vessels surrounded by alveoli. (B) Lungs from OVA-induced mice. Infiltrating inflammatory cells, predominantly eosinophils, were accumulating in the interstitium surrounding the blood vessels and airways. Hyperplasia and disorders of the airway epithelium and shedding of cilia from the epithelial surface were also observed. (C) Lungs in mice that were injected with genistein, showing that damage was markedly reduced. Scale bars, 100 µm. OVA, ovalbumin. Magnification, ×200.

Figure 2.

Airway hyper-responsiveness in response to methacholine administration in mice. Plethysmography was performed 24 h following the final treatment and nebulization. Increasing doses of methacholine were administered and maximal resistance values (cm H₂O/ml/sec) were recorded after 1 min. The ovalbumin-immunized mice exhibited elevated airway resistance with all methacholine doses compared with control and genistein mice (n=6/group). *P<0.05 vs. control and genistein groups. OVA, ovalbumin-immunized group; genistein, genistein-treated group; Re, responsiveness.

Figure 3.

Expression levels of Runx3 mRNA and protein in ovalbumin-immunized and exposed mice. Relative mRNA expression was measured using real-time polymerase chain reaction with SYBR-Green. (A) Runx3 mRNA relative to murine β-actin in lung tissue of ovalbumin-immunized mice compared with that in normal controls and genistein-injected mice (n=8/group). (B) Runx3 protein expression was measured in lung tissue from control, OVA and genistein-injected mice (n=6/group). (C) Nuclear expression of Runx3 protein was determined in lung tissue from control, OVA and genistein-injected (n=6/group) mice. *P<0.05 vs. Control group; **P<0.05 vs. OVA group. Runx3, runt-related transcription factor 3; OVA, ovalbumin-immunized group; Genistein, genistein-treated group.

Figure 4.

Inhibition of ITK affects the expression of Runx3 mRNA and protein. (A) Runx3 mRNA in splenic mononuclear cells in the control, OVA and ITK inhibition groups (n=8/group). (B) Total Runx3 protein expression was detected in the splenic mononuclear cells of the control, OVA, and ITK-inhibition groups (n=6/group). (C) Nuclear expression of Runx3 was detected in the splenic mononuclear cells of the control, OVA and ITK-inhibition groups (n=6/group). *P<0.05 vs. Control group; **P<0.05 vs. OVA group. ITK, inducible T-cell kinase; Runx3, runt-related transcription factor 3; OVA, ovalbumin-immunized group.

Figure 5.

5-Aminomethyl(benzimidazole) is able to activate IFN-γ and silence IL-4 expression in vitro. (A) An increase was observed in supernatant fluid IL-4 in the OVA group compared with the control group and a decrease was observed in the ITK inhibition group compared with the OVA group. (B) A decrease was observed in supernatant fluid IFN-γ in the OVA group compared with the control group and an increase was observed in the ITK inhibition group compared with the OVA group (n=5/group). *P<0.01 vs. Control group; **P<0.05 vs. OVA group. IL, interleukin; IFN, interferon; OVA, ovalbumin-immunized group.