Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac Exchange Factor 1 (p‐rex1) Exacerbates Asthmatic Airway Smooth Muscle Cell Proliferation Via Rac1‐dependent Signal Pathways

Background and Objective Human airway smooth muscle (HASM) hyperplasia and remodeling are characteristic features of airways that contribute to their irreversible obstruction in patients with severe asthma. Accumulating evidence suggests that cell proliferation contributes to airway smooth muscle hyperplasia and remodeling. Although the role of HASM hyperplasia and remodeling in asthma is well established, the molecular mechanisms regulating the proliferation of HASM cells in asthma‐related conditions are poorly understood. P‐Rex1, a Rac‐selective guanine nucleotide exchange factor (GEF), plays important roles in both normal physiology and pathological conditions. The objective of our study was to define the pathological importance and mechanisms of upregulated P‐Rex1 in development of ASM hyperplasia and remodeling of severe asthma. Methods We used immunohistochemistry to compare P‐Rex1 protein expression in lung tissues between human asthmatic and control subjects. Western blot was used to determine changes in P‐Rex1 expression in primary HASM cells isolated from asthmatic and non‐asthmatic subjects. P‐Rex1 expression levels in HASM cells were manipulated by overexpression of recombinant P‐Rex1 or its specific siRNA. The proliferation rate of HASM cells was measured by 5‐Bromo‐2‐deoxyUridine (BrdU) assays. Results Immunohistochemistry staining revealed that P‐Rex1 was expressed in the epithelial layer of the bronchioles. Positive staining was also observed in HASM cells. However, samples from asthmatic subjects had a 4‐fold higher density of P‐Rex1 protein compared to those from control subjects (p < 0.01). Western blot assay also showed 3‐fold higher expression of P‐Rex1 protein in asthmatic HASM cells as compared to non‐asthmatic HASM cells (p<0.01). Overexpression of P‐Rex1 in non‐asthmatic HASM cells increased platelet‐derived growth factor (PDGF)‐stimulated cell proliferation by more than 2‐fold. Conversely, silencing P‐Rex1 in asthmatic HASM cells reduced PDGF‐stimulated cell proliferation by over 70%. Interestingly, treatment with Rac1 inhibitor NSC23766 not only significantly inhibited PDGF‐stimulated cell proliferation in 4 different asthmatic HASM cells but also largely abolished recombinant P‐Rex1 augment of proliferation in non‐asthmatic HASM cells. Conclusion Increase of P‐Rex1 protein promotes HASM cell proliferation via Rac1‐dependent pathways, which in turn causes HASM hyperplasia and remodeling, leading to irreversible airway obstruction. Support or Funding Information This work was supported by grants from the National Institutes of Health (5P20GM103489 and R01HL116849) and Nebraska State LB595 research program