Upregulated P-Rex1 Exacerbates Human Airway Smooth Muscle Hyperplasia in Asthma

Patients with severe asthma often develop poorly reversible airway obstruction. Although various factors such as abnormal airway smooth muscle (ASM) relaxation may contribute to fixed airway obstruction, development of structural changes due to airway remodeling is commonly associated with this complication and poor clinical outcomes in patients.1King G.G. James A. Harkness L. Wark P.A.B. Pathophysiology of severe asthma: we’ve only just started.Respirology. 2018; 23: 262-271Crossref PubMed Scopus (52) Google Scholar Human airway smooth muscle (HASM) hyperplasia is a major component of airway remodeling, and cell proliferation plays an important role in HASM hyperplasia and remodeling.2Berair R. Saunders R. Brightling C.E. Origins of increased airway smooth muscle mass in asthma.BMC Med. 2013; 11: 145-150Crossref PubMed Scopus (54) Google Scholar However, the molecular mechanisms regulating HASM cell proliferation in asthma are still poorly understood. Platelet-derived growth factor (PDGF) is greatly increased in the lungs of patients with asthma and is thought to promote proliferation of HASM cells via activation of tyrosine kinase receptor PDGFR. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase that is required for PDGF-stimulated HASM cell proliferation.3Simeone-Penney M.C. Severgnini M. Rozo L. Takahashi S. Cochran B.H. Simon A.R. PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1.Am J Physiol Lung Cell Mol Physiol. 2008; 294: 698-704Crossref PubMed Scopus (66) Google Scholar However, little is known about how Rac1 activity is regulated in asthmatic HASM cells. P-Rex1 is a Rac-selective guanine nucleotide exchange factor (Rac-GEF) that catalyzes inactive GDP-bound Rac into active GTP-bound form.4Welch H.C.E. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Bromage H.E. et al.P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar We previously reported that aberrantly upregulated P-Rex1 promotes cancer metastasis by activating Rac1 signals.5Qin J. Xie Y. Wang B. Hoshino M. Wolff D.W. Zhao J. et al.Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.Oncogene. 2009; 28: 1853-1863Crossref PubMed Scopus (95) Google Scholar Our report also identified the importance of P-Rex1 in cancer cell proliferation.6Wong C.Y. Jiang H. Abel P.W. Scofield M.A. Xie Y. Wei T. et al.Phorbol myristate acetate suppresses breast cancer cell growth via down-regulation of P-Rex1 expression.Protein Cell. 2016; 7: 445-449Crossref PubMed Scopus (8) Google Scholar P-Rex1 cooperates with PDGFR to drive migratory and invasive phenotypes of cancer cells,7Campbell A.D. Lawn S. McGarry L.C. Welch H.C. Ozanne B.W. Norman J.C. P-Rex1 cooperates with PDGFRβ to drive cellular migration in 3D microenvironments.PLoS One. 2013; 8: e53982Crossref PubMed Scopus (27) Google Scholar but its role in PDGF-stimulated HASM cell proliferation is unknown. Immunohistochemistry staining of formalin-fixed, paraffin-embedded lung samples from subjects with asthma and normal subjects (see Table E1 in this article's Online Repository at www.jacionline.org) revealed that P-Rex1 protein was only weakly expressed in epithelial layers and ASM cells of bronchioles in normal subjects (Fig 1, A, a and b), whereas patients with asthma had a much higher staining density of P-Rex1 protein (Fig 1, A, c and d). Quantitative analysis of lung tissue samples using a ‘‘histo-score’’ (H-score)5Qin J. Xie Y. Wang B. Hoshino M. Wolff D.W. Zhao J. et al.Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.Oncogene. 2009; 28: 1853-1863Crossref PubMed Scopus (95) Google Scholar showed that P-Rex1 protein expression in ASM bundles of patients with asthma was 4-fold higher than that in normal subjects. In contrast, P-Rex-2, an isoform of P-Rex1, was barely detectable in ASM bundles and quantitative analysis showed no significant difference between normal subjects and patients with asthma (see Fig E1 in this article's Online Repository at www.jacionline.org). P-Rex1 upregulation was further confirmed in 3 primary ASM cell lines derived from subjects with asthma, HASM-A, as compared with 3 primary cell lines isolated from subjects without asthma, HASM-N (see Table E2 in this article's Online Repository at www.jacionline.org and Fig 1, B, P < .01). 5-Bromo-2′-deoxyuridine cell proliferation assays indicated that the stimulatory effects of PDGF in asthmatic HASM-A cells are 2-fold higher than that in HASM-N cells (23.2 ± 2.3-fold vs 10.9 ± 1.8-fold, P < .01). Treatment with Rac1 inhibitor NSC23766 (50 μM) had no significant effects on HASM-N cell proliferation, but attenuated PDGF-stimulated proliferation of all 3 HASM-A cell lines by 40% (Fig 2, A) (P < .05), suggesting that active Rac1 promotes asthmatic HASM proliferation.Fig 2Upregulated P-Rex1 exacerbates asthmatic HASM hyperplasia. A, Serum-starved HASM cells were stimulated with 10 ng/mL of PDGF in the absence and presence of 50 μM Rac1 inhibitor NSC23766 for 36 hours, and the cell proliferation was assessed using a BrdU incorporation assay. Inset: Western blot analysis of PDGFR expression levels in 3 nonasthmatic HASM-N1-3 and 3 asthmatic HASM-A1-3 cell lines. B, Nonasthmatic HASM-N cell lines were transfected with empty vector or vector encoding recombinant P-Rex1. C, Asthmatic HASM-A cell lines were transfected with scramble or P-Rex1–specific siRNA using lipofectamine RNAiMAX. Cells were treated with 10 ng/mL of PDGF for 24 hours and then subjected to Western blot analysis of P-Rex1 and PDGFR protein expression (insets) or BrdU incorporation assay. Quantitative analysis of percent BrdU-positive cells was performed by counting BrdU-positive cells compared with total DAPI-stained cells. BrDU, 5-Bromo-2′-deoxyuridine; DAPI, 4′-6-diamidino-2-phenylindole, dihydrochloride. Data are means ± SE from at least 100 cells. All experiments were independently repeated 3 times. *P < .05 and **P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blot analysis indicated that PDGFR expression levels were similar comparing nonasthmatic and asthmatic HASM cell lines (Fig 2, A, inset) and no association was found between PDGFR expression levels and PDGF-stimulated HASM cell proliferation. We next examined whether upregulated P-Rex1 is responsible for asthmatic HASM cell hyperplasia. As shown in Fig 2, B (left panel), overexpression of recombinant P-Rex1 had no significant effects on the basal proliferation rate but significantly increased PDGF-stimulated proliferation of all 3 nonasthmatic HASM-N cell lines. The basal proliferation rate of HSAM-N1 cells transfected with vector or recombinant P-Rex1 was 2.0% ± 0.6% and 2.5% ± 0.3%, respectively. PDGF stimulation increased this value to 7.3% ± 0.1% and 13.1% ± 0.4%, respectively. Similar results were obtained in 2 other HASM-N cell lines (N2 and N3). On average, overexpression of P-Rex1 increased PDGF-stimulated cell proliferation of 3 HASM-N cell lines by 1.9-fold (P < .05) (Fig 2, B, right panel). P-Rex1–specific siRNA5Qin J. Xie Y. Wang B. Hoshino M. Wolff D.W. Zhao J. et al.Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.Oncogene. 2009; 28: 1853-1863Crossref PubMed Scopus (95) Google Scholar was used to reduce P-Rex1 protein levels in 3 asthmatic HASM-A cell lines (Fig 2, C, inset). Silencing endogenous P-Rex1 had little effects on basal proliferation rate but significantly attenuated PDGF-stimulated cell proliferation of all 3 asthmatic HASM-A cell lines (Fig 2, C, left panel). Treatment with PDGF increased the proliferation rate of HASM-A1 cells with scramble siRNA from 3.2% ± 0.5% to 29.7% ± 3.1 %, whereas the proliferation rate of HASM-A1 cells with P-Rex1 siRNA was only increased from 3.3% ± 0.9% to 14.2% ± 2.1% (Fig 2, C, left panel). These results were confirmed in 2 other asthmatic HASM-A cell lines (A2 and A3). On average, knockdown of P-Rex1 expression by 70% to 80% attenuated PDGF-stimulated cell proliferation of 3 HASM-A cell lines by 45.0% ± 3.8% (P < .05) (Fig 2, C, right panel), indicating that upregulated P-Rex1 is required for PDGF-stimulated proliferation of asthmatic HASM-A cells. We also examined the importance of P-Rex1 in epidermal growth factor (EGF)-stimulated HASM cell proliferation (see Fig E2 in this article's Online Repository at www.jacionline.org). The stimulatory effect of EGF in asthmatic HASM-A1 cells was 2.5-fold higher than that in nonasthmatic HASM-N1 cells. P-Rex1 overexpression in HASM-N1 cells increased EGF-stimulated cell proliferation by 2-fold (7.1% ± 0.6% vs 15.6% ± 1.9%), whereas silencing P-Rex1 in HASM-A1 cells reduced EGF-stimulated cell proliferation from 17.5% ± 2.2% to 4.2% ± 1.1%. It should be noted that manipulations of P-Rex1 had little effect on PDGFR or EGF receptor expression in HASM cells (Figs 2 and E2, insets). In conclusion, our results in human tissues and primary cell lines demonstrate an aberrant upregulation of P-Rex1 in HASM of patients with asthma. These data support a key role of P-Rex1 in the increased proliferation of asthmatic HSAM cells, which is likely an essential component regulating ASM cell hyperplasia in asthma. Interestingly, activation of Rac1 by bronchoconstrictor G-protein–coupled receptor ligands in ASM cells plays a major role in airway hyperresponsiveness (AHR), the hallmark of asthma.8André-Grégoire G. Dilasser F. Chesné J. Braza F. Magnan A. Loirand G. et al.Targeting of Rac1 prevents bronchoconstriction and airway hyperresponsiveness.J Allergy Clin Immunol. 2018; 142: 824-833Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Because P-Rex1 is the only Rac-GEF synergistically activated by G-protein–coupled receptors and tyrosine kinase receptors,4Welch H.C.E. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Bromage H.E. et al.P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar upregulated P-Rex1 could also contribute to development of AHR. However, we are aware of some limitations of our study. Rac1 inhibitor NSC23766 at 100 μM exhibits critical off-target effects in vivo.9Dütting S. Heidenreich J. Cherpokova D. Amin E. Zhang S.C. Ahmadian M.R. et al.Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets.J Thromb Haemost. 2015; 13: 827-838Crossref PubMed Scopus (52) Google Scholar Thus, the concentration used in our studies (50 μM) cannot exclude Rac1-independent effects in asthmatic HASM cells. Further research is warranted to determine the functions and molecular mechanisms of upregulated P-Rex1 in asthma. Nonetheless, our data support the concept that P-Rex1 may represent a key target for treating both ASM hyperplasia and AHR, providing a novel approach to reverse airway remodeling and restore airway function in severe asthma. Archived formalin-fixed, paraffin-embedded lung tissue blocks of subjects with asthma (n = 3) and healthy controls (n = 3) were from Dr Reynold A. Panettieri. These tissue samples were obtained during open lung resection from patients at the time of death (Table E1).E1Xie Y. Jiang H. Nguyen H. Jia S. Berro A. Panettieri Jr., R.A. et al.Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness.J Allergy Clin Immunol. 2012; 130: 968-976Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Tissue sections were stained with the HPA001927 anti–P-Rex1 antibody (MilliporeSigma, Burlington, Mass) or anti–P-Rex2 antibody (Cat. NBP1-84275, Novus Biologicals, Littleton, Colo), counterstained with hematoxylin. The negative control used nonimmune rabbit IgG as the primary antibody. The intensity of P-Rex1 protein staining was determined as an average optical density by Image-Pro Plus in 5 randomly chosen airways for each sample. A ‘‘histo-score’’ (H-score) was calculated by multiplying the percentage (P) of positive cells by the average intensity (I) as described previously.E2Qin J. Xie Y. Wang B. Hoshino M. Wolff D.W. Zhao J. et al.Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.Oncogene. 2009; 28: 1853-1863Crossref PubMed Scopus (110) Google ScholarTable E1Subject profiles of archived formalin-fixed, paraffin-embedded lung tissue blocksProfileTissue case numberCause of deathPulmonary diseaseCancerMedicationNonasthma062810Cerebral vascular accidentNoneNoneHypertension medicationN19CMHead gunshot woundNoneNoneNone090710Overdose on Tylenol-intentionalNoneNoneXanax, LisinoprilAsthmaAs081610Anoxia and asthma attackAsthmaNoneAdvair, CombiventAs31BFAnoxia and asthma attackAsthmaNoneAlbuterolAs0726Anoxia and asthma attackAsthmaNoneSingulair Open table in a new tab HASM cells from donors with asthma (A1-3) and donors without asthma (N1-3) were established by Dr Rey Panettieri's laboratory using human lung tissue samples obtained from subjects without and with asthma during open lung resection at the time of death (Table E2). The samples had no identifiers, and the protocol for cell isolation was previously approved by the University of Pennsylvania Institutional Review Board.E3Banerjee A. Trivedi C.M. Damera G. Jiang M. Jester W. Hoshi T. et al.Trichostatin A abrogates airway constriction, but not inflammation, in murine and human asthma models.Am J Respir Cell Mol Biol. 2012; 46: 132-138Crossref PubMed Scopus (58) Google Scholar Cells were cultured at 37°C with 5% CO2 in 1:1 mixture of Dulbecco modified Eagle medium (GE, Logan, Utah) and Ham's F-12 Nutrient Mixture (F12) (Corning, Va) supplemented with 10% FBS and were used at passage less than 10 for experiments.Table E2Subject profiles of isolated nonasthmatic and asthmatic HASM cellsProfileCellCause of deathPulmonary diseaseCancerMedicationNonasthmaN1Gunshot wound neckNoneNoneNoneN2Head/chest trauma caused by vehicle accidentNoneNoneNoneN3Anoxic brain injuryNoneNoneNAAsthmaA1Anoxia and asthma attackAsthmaNoneAlbuterol, AdvairA2Anoxia and asthma attackAsthmaNoneAlbuterolA3Anoxia and asthma attackAsthmaNoneAlbuterolNA, Not applicable. Open table in a new tab NA, Not applicable. Nonasthmatic HASM-N cells (2.5 × 105) were electroporated with 2 μg of either pcDNA3.1 control vector or vector encoding P-Rex1 using the Nucleofector system as per manufacturer's protocol. Transfected cells were cultured for 24 hours and then reseeded for proliferation assays or Western blot analysis as described below. Asthmatic HASM-A cells (2.5 × 105) were transfected with 50 μM of scramble siRNA (negative control #1 siRNA, Ambion, Austin, Tex) or synthesized P-Rex1 siRNA (target sequence of human P-Rex1 gene: 5′-GCAACGACTTCAAGCTGGTGGAGAA-3′)E2Qin J. Xie Y. Wang B. Hoshino M. Wolff D.W. Zhao J. et al.Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.Oncogene. 2009; 28: 1853-1863Crossref PubMed Scopus (110) Google Scholar using lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, Calif) as per manufacturer's protocol. The final concentration of siRNA was 50 nM. Transfected cells were cultured for 24 hours and then reseeded for proliferation assays or Western blot analysis. Cell protein extraction and Western blot were conducted as described previously.E4Xie Y. Jiang H. Zhang Q. Mehrotra S. Abel P.W. Toews M.L. et al.Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis.Respir Res. 2016; 17: 103Crossref PubMed Scopus (20) Google Scholar Protein was extracted from cells using 1× RIPA lysis buffer (Santa Cruz, Dallas, Tex). Samples were electrophoresed and subjected to Western blot using primary antibodies against P-Rex1 (mouse, clone 6F12, MilliporeSigma), PDGF receptor (Cat.3162S, Cell Signaling Technology, Danvers, Mass), EGF receptor (Cat.06-847, MilliporeSigma), and the loading control β-actin (Santa Cruz). IRdye700- or IRdye800-labeled secondary antibodies (LI-COR Biosciences, Lincoln, Neb) were used for protein band detection. The images were captured with a LI-COR Odyssey infrared imaging system (LI-COR Biosciences). For the quantification of the Western blot results, the intensity of each band was divided by the β-actin intensity of that same sample and subsequently divided by the corrected intensity of the untreated sample of the same blot. HASM cells were seeded on coverglasses in 12-well plates (5 × 103 cells/well) and cultured in serum-free medium for 24 hours. Serum-starved HASM cells were stimulated with PDGF-BB (10 ng/mL) or EGF (100 ng/mL) for 24 hours. To test the effect of a Rac1 inhibitor on HASM cells, 50 μM Rac1 inhibitor NSC 23766 (Tocris, Minneapolis, Minn) was added to serum-starved HASM cells 1 hour before PDGF-BB or EGF treatment. Then, 10 μM 5-bromo-2′-deoxyuridine (BrdU) (BD Biosciences, San Jose, Calif) was added to cultured cells for 18 hours. Cells were fixed with 70% ethanol, permeabilized, blocked, and then stained for nuclei and BrdU using 4′,6-diamidino-2-phenylindole, dihydrochloride and anti-BrdU antibody (Cell Signaling Technology). BrdU incorporation into newly synthesized DNA, as an indicator of cell proliferation, was measured by fluorescence microscopy. Results are expressed as the percentage of 4′,6-diamidino-2-phenylindole, dihydrochloride–stained cells that were also BrdU-positive. Data are expressed as means ± SE. Groups were compared using a Student t test for unpaired observations. A probability level (P) of less than .05 was considered statistically significant .Fig E2Upregulated P-Rex1 exacerbates EGF-induced HASM cell proliferation. Nonasthmatic HASM-N1 cells were transfected with empty vector or vector encoding recombinant P-Rex1 (left panel), whereas asthmatic cell HASM-A1 was transfected with scramble or P-Rex1–specific siRNA (right panel) for 48 hours. Cells were treated with 100 ng/mL EGF for 24 hours and then subjected to Western blot analysis of P-Rex1 and EGFR proteins (insets) or BrdU incorporation assay. Quantitative analysis of BrdU-positive cells was performed by counting the percentage of BrdU-positive cells compared with total DAPI-stained cells. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride; EGFR, epidermal growth factor receptor. Data are means ± SE from at least 100 cells. All experiments were independently repeated 3 times. *P < .05 and **P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)