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. 2013 Oct;27(10):3991-4003.
doi: 10.1096/fj.12-221341. Epub 2013 Jun 11.

Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity

Thai Tran  1 , Chun Ming Teoh, John Kit Chung Tam, Yongkang Qiao, Chin Yein Chin, Oi Khuan Chong, Alastair G Stewart, Trudi Harris, Wai Shiu Fred Wong, Shou Ping Guan, Bernard P Leung, William T Gerthoffer, Helmut Unruh, Andrew J Halayko
Affiliations

Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity

Thai Tran et al. FASEB J. 2013 Oct.

Abstract

Increased airway smooth muscle (ASM) mass is believed to underlie the relatively fixed airway hyperresponsiveness (AHR) in asthma. Developments of therapeutic approaches to reverse airway remodeling are impeded by our lack of insight on the mechanisms behind the increase in mass of contractile ASM cells. Increased expression of laminin, an extracellular matrix protein, is associated with asthma. Our studies investigate the role of laminin-induced ASM survival signals in the development of increased ASM and AHR. Antagonizing laminin integrin binding using the laminin-selective competing peptide, YIGSR, and mimicking laminin with exogenous α2-chain laminin, we show that laminin is both necessary and sufficient to induce ASM cell survival, concomitant with the induction of ASM contractile phenotype. Using siRNA, we show that the laminin-binding integrin α7β1 mediates this process. Moreover, in laminin-211-deficient mice, allergen-induced AHR was not observed. Notably, ASM cells from asthmatic airways express a higher abundance of intracellular cell survival proteins, consistent with a role for reduced rates of cell apoptosis in development of ASM hyperplasia. Targeting the laminin-integrin α7β1 signaling pathway may offer new avenues for the development of therapies to reduce the increase in mass of contractile phenotype ASM cells that underlie AHR in asthma.

Keywords: apoptosis; integrin; phenotype plasticity.

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Conflict of interest statement

The authors thank Ms. Karol McNeill and Ms. Tze Khee Chan (TUNEL assay) for excellent technical assistance.

Figures

Figure 1.
Figure 1.
Laminin is required for ASM phenotype maturation and cell survival. A–C) Human ASM cells were treated in the absence or presence of selective laminin-competing peptide YIGSR and then serum deprived for 7 d before protein abundance for sm-α-actin (A), caspase 3 activity (B), and protein abundance for PARP (C) were determined. Results are representative of 3 independent experiments. Data are expressed as fold increment over basal (d 0) relative to β-actin protein abundance. *P < 0.05 vs. d 0; †P < 0.05 vs. d 7 response in absence of peptide. D) Representative immunofluoresence imaging of human ASM cells labeled for sm-α-actin (green) at d 0 and after 7-d serum deprivation (d 7). Nuclei were labeled with Hoechst dye (blue). Cells with condensed or fragmented nuclei (indicated by asterisks) are considered apoptotic. Scale bar = 50 μm. E) Representative TUNEL photographs of human ASM cells at d 0 and after 7-d serum deprivation (d 7). Cells treated with staurosporine (0.1 μm, 7 h) were used as positive control for apoptosis. Cells incubated with labeling solution only (without TdT) were used as negative control. Cells at d 7 show limited TUNEL-positive staining, as indicated by asterisks. Scale bar = 20 μm. F) Representative blots showing effect of sm-α-actin protein abundance and corresponding caspase 3 activity following cotreatment with staurosporine (St; 10−7 M) at the time of serum deprivation. G) Representative blots showing effect of sm-α-actin protein abundance and corresponding caspase 3 activity with staurosporine (St; 10−7 M, 10−6 M) added 3 d after 7-d serum deprivation.
Figure 1.
Figure 1.
Laminin is required for ASM phenotype maturation and cell survival. A–C) Human ASM cells were treated in the absence or presence of selective laminin-competing peptide YIGSR and then serum deprived for 7 d before protein abundance for sm-α-actin (A), caspase 3 activity (B), and protein abundance for PARP (C) were determined. Results are representative of 3 independent experiments. Data are expressed as fold increment over basal (d 0) relative to β-actin protein abundance. *P < 0.05 vs. d 0; †P < 0.05 vs. d 7 response in absence of peptide. D) Representative immunofluoresence imaging of human ASM cells labeled for sm-α-actin (green) at d 0 and after 7-d serum deprivation (d 7). Nuclei were labeled with Hoechst dye (blue). Cells with condensed or fragmented nuclei (indicated by asterisks) are considered apoptotic. Scale bar = 50 μm. E) Representative TUNEL photographs of human ASM cells at d 0 and after 7-d serum deprivation (d 7). Cells treated with staurosporine (0.1 μm, 7 h) were used as positive control for apoptosis. Cells incubated with labeling solution only (without TdT) were used as negative control. Cells at d 7 show limited TUNEL-positive staining, as indicated by asterisks. Scale bar = 20 μm. F) Representative blots showing effect of sm-α-actin protein abundance and corresponding caspase 3 activity following cotreatment with staurosporine (St; 10−7 M) at the time of serum deprivation. G) Representative blots showing effect of sm-α-actin protein abundance and corresponding caspase 3 activity with staurosporine (St; 10−7 M, 10−6 M) added 3 d after 7-d serum deprivation.
Figure 2.
Figure 2.
Laminin is sufficient to induce ASM cell survival signaling. A–C) Human ASM cells were seeded onto plastic or laminin-coated plates and then serum deprived for 7 d before protein abundance for sm-α-actin (A), caspase 3 activity (B), and protein abundance for PARP (C), were determined. α2, affinity-purified α2-chain laminin from human placenta, which includes laminin-211 and -221; EHS, laminin from Engelbreth-Holm-Swarm murine sarcoma consisting mainly of laminin-111. Data are expressed as fold increment over basal (d 0) relative to β-actin protein abundance. *P < 0.05 vs. d 0; †P < 0.05 vs. d 7 response in the absence of laminin coating. D) Representative blots showing effect of α2-chain laminin siRNA on sm-α-actin protein abundance and corresponding caspase 3 activity. TA served as vehicle control. NT, nontargeting siRNA; LN, laminin. Results are representative of 3 independent experiments.
Figure 3.
Figure 3.
Laminin is sufficient to promote ASM cell survival via integrin α7β1. Human ASM cells were treated in the absence or presence of integrin α7 siRNA and then serum-deprived for 6 d before protein abundance for sm-α-actin (A) and integrin α7 (B), caspase 3 activity (C), and protein abundance for PARP (D) were determined. Experiments were terminated following 6-d serum deprivation based on preliminary experiments, which showed effective silencing of integrin α7 mRNA and protein expression for ≥6 d. GFP served as negative control; TA served as vehicle control. Results are representative of 3 independent experiments. Data are expressed as fold increment over basal (d 0) relative to β-actin protein abundance. *P < 0.05 vs. d 0; †P < 0.05 vs. d 6 response without integrin α7 siRNA.
Figure 4.
Figure 4.
Bcl-2 but not Bax is involved in regulating laminin-induced ASM cell survival. Bcl-2 (A–C) or Bax (D–F) protein abundance was measured following 7- or 6-d serum deprivation in the presence or absence of selective laminin-competing peptide YIGSR (A, D), various types of laminin coating (B, E) or integrin α7 siRNA (C, F). Experiments were terminated after 6-d serum deprivation based on preliminary experiments, which showed effective silencing of integrin α7 mRNA and protein expression for ≥6 d for siRNA experiments. Results are representative of 3 independent experiments. α2, affinity-purified α2-chain laminin from human placenta, which includes laminin-211 and -221; EHS, laminin from EHS murine sarcoma consisting mainly of laminin-111. GFP served as negative control; TA served as vehicle control. Data are expressed as fold increment over basal (d 0) relative to β-actin protein abundance. *P < 0.05 vs. d 0; †P < 0.05 vs. d 7 response in absence of peptide or laminin coating or d 6 response without integrin α7 siRNA.
Figure 5.
Figure 5.
PI3K links ASM contractile phenotype signaling with cell survival. A) Effect of PI3K inhibitors, LY294002 (LY, 20 μM) and wortmannin (W, 100 nM), on the protein abundance of Akt, sm-α-actin, and Bcl-2. LYD and WD are vehicle controls for LY294002 (0.2% DMSO) and wortmannin (0.01% DMSO), respectively. B) Effect of selective laminin-competing peptide YIGSR on the protein abundance of Akt. Results are representative of 3 independent experiments.
Figure 6.
Figure 6.
Asthmatic primary human ASM cells express higher levels of cell survival proteins than nonasthmatic primary human ASM cells. Comparison of the protein abundance levels for sm-α-actin, Bcl-2, Bax, and α2-chain laminin (LN) (A), and caspase 3 activity between primary cultured human ASM cells from asthmatic vs. healthy nonasthmatic airways (B). Results are representative of 4 independent experiments. *P < 0.05 vs. d 0 nonasthmatic.
Figure 7.
Figure 7.
Lama2−/− mice do not develop allergen challenge-induced airway hyperreactivity. A) Comparison of airway reactivity between wild-type (WT) and Lama2−/− mice (n=4–7/group); results for Lama2−/− saline sensitization and saline challenge (s/s) and Lama2−/− OVA sensitization and OVA challenge (o/o) are superimposed. *P < 0.05 vs. WT (s/s)-treated mice; †P < 0.05 vs. WT (o/o). B) Comparison of caspase 3 activity between WT and Lama2−/− mice (n=3–4/group). *P < 0.05 vs. WT (s/s)-treated mice. C) Comparison of sm-α-actin-positive area relative to basement membrane (BM) between WT and Lama2−/− mice (n=3–4/group). *P < 0.05 vs. WT (s/s); †P < 0.05 vs. WT (o/o). D) H&E and immunohistochemistry staining for sm-α-actin and TUNEL staining in mouse whole-lung slices. Asterisks indicate TUNEL-positive apoptotic cells. Scale bars = 20 μm. E–G) Levels of OVA-specific IgE (E), IgG1 (F), and IgG2a (G) in mouse serum that was collected 24 h after the last OVA aerosol challenge. O.D., optical density. *P < 0.05 vs. WT (s/s)-treated mice.
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