Pim1 inactivating induces RUNX3 upregulation that improves/alleviates airway inflammation and mucus hypersecretion in vitro and in vivo

Abstract

Purpose: Our research aimed to evaluate whether proto-oncogene serine/threonine-protein kinase Pim-1 (Pim1) inactivation could attenuate asthma by promoting runt-related transcription factor 3 (Runx3) expression and explore the underlying molecular mechanism.

Method: Phorbol 12-myristate 13-acetate (PMA, 50 nM) was used to induce inflammation in BEAS-2B human airway epithelial cells. ELISA and immunofluorescence double staining confirmed inflammation modelling and differential expression of Pim1 and Runx3. Pim1 inhibitor (SGI-1776) and Runx3 siRNA (siRunx3) were used in this study. Apoptosis, inflammation, MUC5AC protein expression, Pim1 kinase and Runx3 protein expression, and PI3K/AKT/nuclear factor-κB (NF-κB) pathway-associated protein expression were also assessed by flow cytometry, immunofluorescence and western blot. The effects of Pim1 inactivation on airway inflammation, pathological injury and mucus secretion in wild-type and Runx3 knockout mice were observed by in vivo experiments.

Results: The results of the in vitro experiments showed that PMA stimulation causes BEAS-2B cell apoptosis and promotes the MUC5AC expression. In addition, PMA stimulation activated the PI3K/AKT/NF-κB pathway. SGI-1776 treatment partially reversed these effects, whereas siRunx3 attenuated the effects of SGI-1776 on PMA-stimulated BEAS-2B cells. In vivo experiments showed that in Runx3-KO asthmatic mice, inhibition of Pim1 kinase had less effect on airway inflammation, pathological injury and mucus secretion. Meanwhile, Pim1 kinase expression was higher in Runx3-KO asthmatic mice than in wild-type asthmatic mice. Furthermore, inhibition of Pim1 kinase inhibited activation of the PI3K/AKT/NF-κB pathway, whereas these effects were attenuated in Runx3-KO mice.

Conclusion: Our results suggest that Pim1 inactivation can ameliorate airway inflammation and mucus hypersecretion through upregulation of Runx3 and the effect could be mediated through modulation of the PI3K/AKT/NF-κB pathway.

Keywords: Airway Epithelium; Asthma.

Figure 1. The localisation and expression of Pim1 and Runx3 in BEAS-2B cells after PMA stimulation. Dual immunofluorescence staining of Pim1/Runx3 was performed. Pim1 (green), RUNX3 (red) and nucleus (blue). The scale bar was 50 µm (the magnification of 200×).

Figure 2. Pim1 inactivation inhibits epithelial cell apoptosis induced by RUNX3. (A) The expression of Pim1 and Runx3 proteins in BEAS-2B cells was detected using western blotting. (B) Cell apoptosis of BEAS-2B cells was detected using flow cytometry. **P <0.05.

Figure 3. Pim1 inactivation decreases inflammatory factor levels and MUC5AC expression in phorbol-12-myristate-13-acetate (PMA)-induced BEAS-2B cells. (A) The levels of interleukin 5 (IL-5), interleukin 13 (IL-13), monocyte chemotactic protein-1 (MCP-1) and human cysteinyl leukotrienes (Cys-LT) were detected by the ELISA method. (B) The expression of MUC5AC was detected using immunofluorescence. **P <0.05.

Figure 4. Pim1 regulated Runx3 via the PI3K/AKT/NF-κB pathway in phorbol-12-myristate-13-acetate (PMA)-induced BEAS-2B cells. The relative expressions of p-PI3K, PI3K, p-Akt, Akt, p-NF-κB, nuclear factor-κB (NF-κB), p-PTEN and PTEN were detected by western blotting. **P <0.05.

Figure 5. Pim1 regulated asthma progression through Runx3. (A) The respiratory resistance value was calculated to evaluate the changes in airway resistance in mice after each methylcholine excitation. (B) The number of total cells, eosinophils, macrophages, neutrophils and lymphocytes in BALF were counted. (C) IgE levels were measured by ELISA. **P <0.05.

Figure 6. Pim1 regulates Runx3 to improve airway inflammation and inhibit mucus secretion via the PI3K/AKT/NF-κB pathway. (A) H&E and periodic acid-Schiff (PAS) staining was used to assess inflammatory cell infiltration and mucus secretion in the airway. Black arrows indicate positive PAS expression. (B) PAS-positive rate. (C) Inflammatory score. (D) The expression of MUC5AC was detected using immunofluorescence. (E) The levels of interleukin 5 (IL-5), interleukin 13 (IL-13), monocyte chemotactic protein-1 (MCP-1) and human cysteinyl leukotrienes (Cys-LT) in BALF were detected by ELISA. **P <0.05.

Figure 7. Pim1 regulates the PI3K/AKT/NF-κB pathway via Runx3. The expression of Pim1, Runx3, p-PI3K, PI3K, p-Akt, Akt, p-NF-κB, nuclear factor-κB (NF-κB), p-PTEN and PTEN was detected by western blotting. ^**P <0.05.