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PNAS 110 (2): 660-665

Copyright © 2013 by the National Academy of Sciences.

Mfge8 suppresses airway hyperresponsiveness in asthma by regulating smooth muscle contraction

Makoto Kudoa,b, S. M. Amin Khalifeh Soltanic,d, Stephen A. Sakumac,d, William McKleroyc,d, Ting-Hein Leec,d, Prescott G. Woodruffd, Jae Woo Leee, Katherine Huanga,d, Chun Chena,d, Mehrdad Arjomandia,f, Xiaozhu Huanga,d, and Kamran Atabaia,c,d,1

aLung Biology Center, University of California, San Francisco, CA 94143; bDepartment of Internal Medicine and Clinical Immunology, Graduate School of Medicine, Yokohama City University, Yokohama 236-0004, Japan; cCardiovascular Research Institute, University of California, San Francisco, CA 94143; dDepartment of Medicine, University of California, San Francisco, CA 94143; eDepartment of Anesthesia, University of California, San Francisco, CA 94143; and fPulmonary Research Group, San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121;


Figure 01
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Fig. 1. Mfge8–/– mice develop exaggerated AHR after Ova sensitization and challenge. (A) Mice were treated with the 36-d Ova protocol as described in Materials and Methods. Mfge8–/– mice developed significantly greater pulmonary resistance to acetylcholine 24 h after the last Ova challenge (n = 9–10 in each experimental group; *P < 0.001, **P < 0.01, ***P < 0.05, Ova-treated Mfge8–/– vs. WT mice). (B) Mice were sensitized with the 70-d Ova protocol as described in Materials and Methods. Mfge8–/– mice developed significantly greater pulmonary resistance to acetylcholine 24 h after the last Ova challenge (n = 18–20 for Ova-treated group and n = 14–20 for saline-treated group; *P < 0.001, **P < 0.01, Ova-treated Mfge8–/– vs. WT mice). (C) Tracheal rings were taken from mice completing the 36-d Ova protocol, and contractile force was measured. Mfge8–/– tracheal rings generated significantly greater contraction to MCh (n = 8 for Ova-treated and n = 4 for saline solution-treated tracheal rings; *P < 0.001, **P < 0.01, Ova-treated Mfge8–/– vs. WT rings). (D) Tracheal rings were treated with IL-13 (100 ng/mL) for 12 h before measuring contraction. Mfge8–/– tracheal rings generated significantly greater contraction to MCh (n = 12; *P < 0.05, **P < 0.001, Ova-treated Mfge8–/– vs. WT rings).

 

Figure 02
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Fig. 2. Mfge8 reduces ASM contraction through its integrin-binding and discoidin domains. (A) IL-13–treated tracheal rings were incubated with rMfge8 for 1 or 12 h before challenge with MCh (n = 5–14; *P < 0.001, IL-13–treated Mfge8–/– rings treated with rMfge8 vs. IL-13–treated rings receiving no additional treatment for 1 h; **P < 0.001, IL-13–treated Mfge8–/– rings treated with rMfge8 vs. IL-13–treated rings receiving no additional treatment for 12 h). There was no significant difference between vehicle-treated Mfge8–/– rings treated with rMfge8 and vehicle-treated rings receiving no additional treatment. (B) IL-13–treated WT tracheal rings generate significantly less contraction when incubated with rMfge8 (n = 6–12; *P < 0.001, **P < 0.01, and ***P < 0.05, IL-13–treated WT rings treated with rMfge8 vs. IL-13–treated rings receiving no additional treatment). (C) Mfge8 mutated protein constructs fused to a human Fc domain. Mfge8 contains two EGF domains (E1 and E2), two discoidin domains (D1 and D2), and a proline-threonine rich mucin-like domain (PT). (D) The full-length construct and a construct lacking the second discoidin domain (E1E2D1) significantly reduced contraction (n = 5–29; *P < 0.001, Mfge8–/–_IL-13_rMfge8 vs. Mfge8–/–_IL-13 treatment; **P < 0.001, ***P < 0.001, Mfge8–/–_IL-13_E1E2D1 construct vs. Mfge8–/–_IL-13 treatment). A one-way ANOVA followed by a Bonferroni t test for subsequent pairwise comparison was used for all statistical analysis. All data are expressed as mean ± SEM.

 

Figure 03
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Fig. 3. Mfge8 regulates calcium sensitivity. (A) Tracheal ring muscle strips were incubated with IL-13 for 12 h and treated with MCh for 15 min, after which protein was extracted for Western blotting and examined with antibodies directed against MLC, pMLC, MYPT1, pMYPT1, GAPDH, RhoA, ROCK1, and ROCK2. For quantification of NF-{kappa}B, the nuclear fraction was probed with antibodies directed against NF-{kappa}B and histone H1 as a loading control. (B) Tracheal ring muscle strips were incubated with IL-13 for 12 h and rMfge8 (10 μg/mL) or rMfge8_RGE (10 μg/mL) for 1 h and treated with MCh for 15 min, and Western blots performed as described in A. (C) Primary ASM isolated from tracheal rings were incubated with IL-13 (100 ng/mL) and rMfge8 (10 μg/mL), rMfge8_RGE (10 μg/mL), or vehicle control for 1 h and stimulated with 5-hydroxytryptamine for 15 min, after which active and total RhoA was evaluated by using a GST pull-down assay and Western blot. (D) Tracheal rings from mice completing the 36-d Ova protocol were incubated with MCh for 15 min and then processed for Western blot as described in A.

 

Figure 04
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Fig. 4. Mfge8 regulates contraction in human ASM, and expression is decreased in human asthmatic patients. (A) Human bronchial rings were treated with IL-13 and lactadherin (10 and 20 μg/mL) for 12 h, and force of contraction was measured (n = 5–10; *P < 0.001, **P < 0.05, IL-13–treated rings vs. lactadherin, one-way ANOVA followed by Bonferroni test for subsequent pairwise comparison). (B) Homogenates from endobronchial biopsies of healthy and asthmatic human subjects were separated by SDS/PAGE and probed for Mfge8, α-smooth muscle actin, and histone H3 as a loading control. (C, D, and E) Blots were subsequently analyzed by densitometry and normalized to loading control pixel density values. Biopsies from asthmatic patients expressed higher levels of α-smooth muscle actin (C) but lower levels of Mfge8 (D; **P < 0.01, *P < 0.001). (E) Expression of Mfge8 was normalized to α-smooth muscle actin. Asthmatic patients expressed less Mfge8 relative to α-smooth muscle actin than healthy controls.

 


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