Limits...
IgE induces proliferation in human airway smooth muscle cells: role of MAPK and STAT3 pathways.

Redhu NS, Shan L, Al-Subait D, Ashdown HL, Movassagh H, Lamkhioued B, Gounni AS - Allergy Asthma Clin Immunol (2013)

Bottom Line: Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function.Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation.Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Faculty of Medicine, University of Manitoba, 419 Apotex Centre- 750 McDermot Ave, Winnipeg, MB R3E 0T5, Canada. abdel.gounni@med.umanitoba.ca.

ABSTRACT
Airway remodeling is not specifically targeted by current asthma medications, partly owing to the lack of understanding of remodeling mechanisms, altogether posing great challenges in asthma treatment. Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function. Recent evidence suggests that IgE sensitization can enhance the survival and mediator release in inflammatory cells. Human ASM (HASM) cells express both low affinity (FcεRII/CD23) and high affinity IgE Fc receptors (FcεRI), and IgE can modulate the contractile and synthetic function of HASM cells. IgE was recently shown to induce HASM cell proliferation but the detailed mechanisms remain unknown. We report here that IgE sensitization induces HASM cell proliferation, as measured by 3H-thymidine, EdU incorporation, and manual cell counting. As an upstream signature component of FcεRI signaling, inhibition of spleen tyrosine kinase (Syk) abrogated the IgE-induced HASM proliferation. Further analysis of IgE-induced signaling depicted an IgE-mediated activation of Erk 1/2, p38, JNK MAPK, and Akt kinases. Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation. Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation.

No MeSH data available.


Related in: MedlinePlus

Lentivirus-mediated STAT3-inhibition abrogates IgE- and PDGF-induced proliferation in HASM cells. (A) IgE-induced STAT3 phosphorylation was determined by Western blotting. (B) Summary of four experiments to assess IgE-induced STAT3 phosphorylation. (C) Expression of total STAT3 in wild-type (WT or non-transduced), scramble-shRNA, and STAT3-shRNA–transduced HASM cells was analyzed by Western blotting. (D) Stably lentivirus-transduced HASM cells were stimulated with IgE, PDGF-BB, or 10% FBS, and 3H-thymidine incorporation was performed. Data represents mean ± SEM; *p < 0.05, ***p < 0.001, ns, non-significant compared with respective unstimulated cells. ###p < 0.001 compared with STAT3-shRNA-transduced unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842672&req=5

Figure 5: Lentivirus-mediated STAT3-inhibition abrogates IgE- and PDGF-induced proliferation in HASM cells. (A) IgE-induced STAT3 phosphorylation was determined by Western blotting. (B) Summary of four experiments to assess IgE-induced STAT3 phosphorylation. (C) Expression of total STAT3 in wild-type (WT or non-transduced), scramble-shRNA, and STAT3-shRNA–transduced HASM cells was analyzed by Western blotting. (D) Stably lentivirus-transduced HASM cells were stimulated with IgE, PDGF-BB, or 10% FBS, and 3H-thymidine incorporation was performed. Data represents mean ± SEM; *p < 0.05, ***p < 0.001, ns, non-significant compared with respective unstimulated cells. ###p < 0.001 compared with STAT3-shRNA-transduced unstimulated cells.

Mentions: STAT3 activation is indispensable in HASM cell proliferation in response to PDGF[8]. Interestingly, monomeric IgE induces STAT3 phosphorylation in murine bone marrow-derived mast cells and rat basophilic leukemia cells, and induce the transcription of genes important in cell survival[24]. With these reports in consideration, we first sought to determine whether IgE is able to phosphorylate STAT3 in HASM cells. A representative blot in Figure 5A and summary of 4 experiments in Figure 5B show that IgE indeed induced STAT3 phosphorylation in HASM cells. To confirm its role in HASM cell proliferation, we employed lentiviral vector-mediated STAT3 silencing approach[20]. HASM cells were stably transduced with pseudotyped lentiviral vector encoding specific STAT3-shRNA. Mock and scramble sequence served as controls. More than 95% of HASM cells were transduced as observed by turbo-GFP signal by FACS analysis (data not shown). Lentiviral-STAT3-shRNA transduction resulted in a noticeable decrease in STAT3 expression compared to WT or scramble-shRNA transduction controls (Figure 5C)[21]. Both scramble-shRNA- and STAT3-shRNA-transduced HASM cells were stimulated with IgE and PDGF to analyze thymidine incorporation. Since PDGF-induced mitogenic signaling requires STAT3 expression[8], 10% FBS was used as an additional positive control in this experiment. As expected, scramble-shRNA-transduced HASM cells showed a normal and statistically significant response to IgE (p < 0.05), PDGF, and 10% FBS (p < 0.001) compared with unstimulated control (Figure 5D). However, the effect of IgE was completely abrogated in STAT3-shRNA transduced cells, and so was the effect of PDGF, also confirming the previous reports[8]. On the other hand, although 10% FBS showed increased thymidine incorporation in STAT3-shRNA-transduced cells, the effect was much less pronounced when compared with scramble-shRNA-transduced HASM cells (Figure 5D). This is consistent with the observation by other groups[8], and suggests that the serum components may also require STAT3 activation to induce mitogenic signaling in HASM cells. In summary, our data suggest that IgE-induced STAT3 activation plays a critical role in HASM cell proliferation.


IgE induces proliferation in human airway smooth muscle cells: role of MAPK and STAT3 pathways.

Redhu NS, Shan L, Al-Subait D, Ashdown HL, Movassagh H, Lamkhioued B, Gounni AS - Allergy Asthma Clin Immunol (2013)

Lentivirus-mediated STAT3-inhibition abrogates IgE- and PDGF-induced proliferation in HASM cells. (A) IgE-induced STAT3 phosphorylation was determined by Western blotting. (B) Summary of four experiments to assess IgE-induced STAT3 phosphorylation. (C) Expression of total STAT3 in wild-type (WT or non-transduced), scramble-shRNA, and STAT3-shRNA–transduced HASM cells was analyzed by Western blotting. (D) Stably lentivirus-transduced HASM cells were stimulated with IgE, PDGF-BB, or 10% FBS, and 3H-thymidine incorporation was performed. Data represents mean ± SEM; *p < 0.05, ***p < 0.001, ns, non-significant compared with respective unstimulated cells. ###p < 0.001 compared with STAT3-shRNA-transduced unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842672&req=5

Figure 5: Lentivirus-mediated STAT3-inhibition abrogates IgE- and PDGF-induced proliferation in HASM cells. (A) IgE-induced STAT3 phosphorylation was determined by Western blotting. (B) Summary of four experiments to assess IgE-induced STAT3 phosphorylation. (C) Expression of total STAT3 in wild-type (WT or non-transduced), scramble-shRNA, and STAT3-shRNA–transduced HASM cells was analyzed by Western blotting. (D) Stably lentivirus-transduced HASM cells were stimulated with IgE, PDGF-BB, or 10% FBS, and 3H-thymidine incorporation was performed. Data represents mean ± SEM; *p < 0.05, ***p < 0.001, ns, non-significant compared with respective unstimulated cells. ###p < 0.001 compared with STAT3-shRNA-transduced unstimulated cells.
Mentions: STAT3 activation is indispensable in HASM cell proliferation in response to PDGF[8]. Interestingly, monomeric IgE induces STAT3 phosphorylation in murine bone marrow-derived mast cells and rat basophilic leukemia cells, and induce the transcription of genes important in cell survival[24]. With these reports in consideration, we first sought to determine whether IgE is able to phosphorylate STAT3 in HASM cells. A representative blot in Figure 5A and summary of 4 experiments in Figure 5B show that IgE indeed induced STAT3 phosphorylation in HASM cells. To confirm its role in HASM cell proliferation, we employed lentiviral vector-mediated STAT3 silencing approach[20]. HASM cells were stably transduced with pseudotyped lentiviral vector encoding specific STAT3-shRNA. Mock and scramble sequence served as controls. More than 95% of HASM cells were transduced as observed by turbo-GFP signal by FACS analysis (data not shown). Lentiviral-STAT3-shRNA transduction resulted in a noticeable decrease in STAT3 expression compared to WT or scramble-shRNA transduction controls (Figure 5C)[21]. Both scramble-shRNA- and STAT3-shRNA-transduced HASM cells were stimulated with IgE and PDGF to analyze thymidine incorporation. Since PDGF-induced mitogenic signaling requires STAT3 expression[8], 10% FBS was used as an additional positive control in this experiment. As expected, scramble-shRNA-transduced HASM cells showed a normal and statistically significant response to IgE (p < 0.05), PDGF, and 10% FBS (p < 0.001) compared with unstimulated control (Figure 5D). However, the effect of IgE was completely abrogated in STAT3-shRNA transduced cells, and so was the effect of PDGF, also confirming the previous reports[8]. On the other hand, although 10% FBS showed increased thymidine incorporation in STAT3-shRNA-transduced cells, the effect was much less pronounced when compared with scramble-shRNA-transduced HASM cells (Figure 5D). This is consistent with the observation by other groups[8], and suggests that the serum components may also require STAT3 activation to induce mitogenic signaling in HASM cells. In summary, our data suggest that IgE-induced STAT3 activation plays a critical role in HASM cell proliferation.

Bottom Line: Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function.Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation.Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Faculty of Medicine, University of Manitoba, 419 Apotex Centre- 750 McDermot Ave, Winnipeg, MB R3E 0T5, Canada. abdel.gounni@med.umanitoba.ca.

ABSTRACT
Airway remodeling is not specifically targeted by current asthma medications, partly owing to the lack of understanding of remodeling mechanisms, altogether posing great challenges in asthma treatment. Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function. Recent evidence suggests that IgE sensitization can enhance the survival and mediator release in inflammatory cells. Human ASM (HASM) cells express both low affinity (FcεRII/CD23) and high affinity IgE Fc receptors (FcεRI), and IgE can modulate the contractile and synthetic function of HASM cells. IgE was recently shown to induce HASM cell proliferation but the detailed mechanisms remain unknown. We report here that IgE sensitization induces HASM cell proliferation, as measured by 3H-thymidine, EdU incorporation, and manual cell counting. As an upstream signature component of FcεRI signaling, inhibition of spleen tyrosine kinase (Syk) abrogated the IgE-induced HASM proliferation. Further analysis of IgE-induced signaling depicted an IgE-mediated activation of Erk 1/2, p38, JNK MAPK, and Akt kinases. Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation. Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation.

No MeSH data available.


Related in: MedlinePlus