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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

IgE-induced HASM cell proliferation requires Syk activity. (A) HASM cells were stably transduced with pseudotyped lentiviral vectors expressing Syk-shRNA or non-specific scramble-shRNA. Syk expression was assessed by Western blotting as described in Material and Methods section. (B) Syk- or scramble-shRNA-transduced HASM cells were stimulated with IgE (10 μg/ml), PDGF-BB (10 ng/ml), or left unstimulated. 3H-thymidine incorporation was measured as a marker of cell proliferation as in Figure 1A. n = 3, *p < 0.05, ***p < 0.001, and ns, non-significant compared to unstimulated control.
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Figure 2: IgE-induced HASM cell proliferation requires Syk activity. (A) HASM cells were stably transduced with pseudotyped lentiviral vectors expressing Syk-shRNA or non-specific scramble-shRNA. Syk expression was assessed by Western blotting as described in Material and Methods section. (B) Syk- or scramble-shRNA-transduced HASM cells were stimulated with IgE (10 μg/ml), PDGF-BB (10 ng/ml), or left unstimulated. 3H-thymidine incorporation was measured as a marker of cell proliferation as in Figure 1A. n = 3, *p < 0.05, ***p < 0.001, and ns, non-significant compared to unstimulated control.

Mentions: FcεRI activation leads to a spectrum of signaling events in inflammatory cells, starting with phosphorylation of Lyn kinase followed by recruitment and phosphorylation of Syk. Activation of Syk then serves as an indispensable mechanism of downstream propagation of signals leading to the activation of various kinases, transcription factors, mediator release, and survival[2,22,23]. This suggests that inhibition/silencing of Syk might be a useful strategy to validate the role of Syk and FcεRI pathway in IgE-induced HASM cell proliferation. To test this, we utilized the lentiviral-mediated Syk inhibition strategy, which we have reported earlier in IgE-induced mediator release in HASM cells[15,17]. HASM cells were stably transduced with pseudotyped lentiviral vector expressing specific Syk-shRNA. Mock and scramble sequence were used as negative controls. As reported earlier[15,17], more than 95% of HASM cells were transduced by turbo-GFP signal positivity by FACS analysis (data not shown). Lentiviral-Syk-shRNA but not control scramble-shRNA transduction resulted in a highly significant and reproducible decrease in Syk expression, as shown by Western blotting (Figure 2A). We then used these lentiviral-transduced cells and stimulated them with IgE and PDGF. As shown in Figure 2B, scramble-shRNA-transduced HASM cells demonstrated a significant increase in thymidine incorporation (p < 0.05, n = 3) similar to the wild-type cells (Figure 1A). However, Syk-shRNA-transduced cells lost the effect of IgE (p > 0.05, non-significant). PDGF consistently showed highly significant (p < 0.001) thymidine incorporation in both scramble and Syk-inhibited HASM cells (Figure 2B). These results suggest that IgE-induced proliferation requires the function of Syk, a key signaling pathway in FcεRI activation.


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)

IgE-induced HASM cell proliferation requires Syk activity. (A) HASM cells were stably transduced with pseudotyped lentiviral vectors expressing Syk-shRNA or non-specific scramble-shRNA. Syk expression was assessed by Western blotting as described in Material and Methods section. (B) Syk- or scramble-shRNA-transduced HASM cells were stimulated with IgE (10 μg/ml), PDGF-BB (10 ng/ml), or left unstimulated. 3H-thymidine incorporation was measured as a marker of cell proliferation as in Figure 1A. n = 3, *p < 0.05, ***p < 0.001, and ns, non-significant compared to unstimulated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: IgE-induced HASM cell proliferation requires Syk activity. (A) HASM cells were stably transduced with pseudotyped lentiviral vectors expressing Syk-shRNA or non-specific scramble-shRNA. Syk expression was assessed by Western blotting as described in Material and Methods section. (B) Syk- or scramble-shRNA-transduced HASM cells were stimulated with IgE (10 μg/ml), PDGF-BB (10 ng/ml), or left unstimulated. 3H-thymidine incorporation was measured as a marker of cell proliferation as in Figure 1A. n = 3, *p < 0.05, ***p < 0.001, and ns, non-significant compared to unstimulated control.
Mentions: FcεRI activation leads to a spectrum of signaling events in inflammatory cells, starting with phosphorylation of Lyn kinase followed by recruitment and phosphorylation of Syk. Activation of Syk then serves as an indispensable mechanism of downstream propagation of signals leading to the activation of various kinases, transcription factors, mediator release, and survival[2,22,23]. This suggests that inhibition/silencing of Syk might be a useful strategy to validate the role of Syk and FcεRI pathway in IgE-induced HASM cell proliferation. To test this, we utilized the lentiviral-mediated Syk inhibition strategy, which we have reported earlier in IgE-induced mediator release in HASM cells[15,17]. HASM cells were stably transduced with pseudotyped lentiviral vector expressing specific Syk-shRNA. Mock and scramble sequence were used as negative controls. As reported earlier[15,17], more than 95% of HASM cells were transduced by turbo-GFP signal positivity by FACS analysis (data not shown). Lentiviral-Syk-shRNA but not control scramble-shRNA transduction resulted in a highly significant and reproducible decrease in Syk expression, as shown by Western blotting (Figure 2A). We then used these lentiviral-transduced cells and stimulated them with IgE and PDGF. As shown in Figure 2B, scramble-shRNA-transduced HASM cells demonstrated a significant increase in thymidine incorporation (p < 0.05, n = 3) similar to the wild-type cells (Figure 1A). However, Syk-shRNA-transduced cells lost the effect of IgE (p > 0.05, non-significant). PDGF consistently showed highly significant (p < 0.001) thymidine incorporation in both scramble and Syk-inhibited HASM cells (Figure 2B). These results suggest that IgE-induced proliferation requires the function of Syk, a key signaling pathway in FcεRI activation.

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