The phosphatase CD148 promotes airway hyperresponsiveness through SRC family kinases

Abstract

Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine phosphorylation is tightly regulated by the opposing actions of protein tyrosine kinases and phosphatases, but little is known about whether tyrosine phosphatases influence AHR. Here, we demonstrate that genetic inactivation of receptor-like protein tyrosine phosphatase J (Ptprj), which encodes CD148, protected mice from the development of increased AHR in two different asthma models. Surprisingly, CD148 deficiency minimally affected the inflammatory response to allergen, but significantly altered baseline pulmonary resistance. Mice specifically lacking CD148 in smooth muscle had decreased AHR, and the frequency of calcium oscillations in CD148-deficient ASM was substantially attenuated, suggesting that signaling pathway alterations may underlie ASM contractility. Biochemical analysis of CD148-deficient ASM revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC family kinases (SFKs), implicating CD148 as a critical positive regulator of SFK signaling in ASM. The effect of CD148 deficiency on ASM contractility could be mimicked by treatment of both mouse trachea and human bronchi with specific SFK inhibitors. Our studies identify CD148 and the SFKs it regulates in ASM as potential targets for the treatment of AHR.

Figure 1. CD148-deficient mice are protected from AHR in the OVA mouse model of allergic airway disease and have decreased baseline pulmonary resistance.

(A) Pulmonary resistance measurements in WT and CD148-deficient (Ptprj^TM–/TM–) mice following immunization and intranasal challenge with OVA or saline in C57BL/6 mice (A) or BALB/c mice (B). Data are the mean ± SEM (n = 9–13 animals per group). Statistical significance determined by 2-way ANOVA. *P < 0.05, ***P < 0.001 in the WT versus Ptprj^TM–/TM– OVA group; ^##P < 0.01, ^###P < 0.001 comparing the highest dose of ACh in the WT saline group with the Ptprj^TM–/TM– saline group.

Figure 2. CD148 deficiency does not attenuate inflammatory response following OVA allergen sensitization and challenge.

(A and D) BAL cell counts of total cells, macrophages, eosinophils, lymphocytes, and neutrophils in WT and CD148-deficient (Ptprj^TM–/TM–) mice following immunization and intranasal challenge with OVA or saline in C57BL/6 (A) or BALB/c (D) mice. (B and E) Histologic scoring on a scale of 0 to 4 by a blinded observer of H&E staining to quantify the degree of airway inflammation (left panel) and PAS staining to quantify mucus-producing goblet cells (right panel) in WT and Ptprj^TM–/TM– mice. (C and F) Relative OVA-specific serum IgE levels were measured by ELISA in WT and Ptprj^TM–/TM– mice. Data for all panels include 9–13 animals per group. Data show the mean ± SEM. *P < 0.05, unpaired 2-tailed Student’s t test. NS, nonsignificant.

Figure 3. CD148 does not regulate T cell immune responses to allergen.

BALB/c mice were immunized and sensitized with OVA, and cells from the lungs and mediastinal lymph nodes were stimulated with PMA and ionomycin and analyzed using intracellular cytokine staining. Total CD4 counts were similar in lung (A) and mediastinal lymph node (C). Absolute numbers of CD4⁺ IL4⁺ (B and D), CD4⁺ IL13⁺ (E and G), CD4⁺ IL17⁺ (F and H), CD4⁺ IFNγ⁺ (I and K), and γδTCR⁺ IL17⁺ (J and L) cells in both lung and lymph node were not statistically significantly different by unpaired 2-tailed Student’s t test. Data are the mean ± SEM for at least 5 mice per group.

Figure 4. CD148-deficient mice are protected from HDM-induced AHR with minimal attenuation of inflammatory response to allergen.

(A) Pulmonary resistance measurements in WT and Ptprj^TM–/TM– BALB/c mice following immunization and intranasal challenge with HDM. ***P < 0.001 for the WT HDM versus the Ptprj^TM–/TM– HDM group; ^###P < 0.001 comparing the highest dose of ACh in the WT saline group with the Ptprj^TM–/TM– saline group by 2-way ANOVA. (B) BAL cell counts of total cells, macrophages, eosinophils, lymphocytes, and neutrophils in WT and Ptprj^TM–/TM– mice. (C) Histologic scoring on a scale of 0 to 4 by a blinded observer of H&E staining to quantify degree of airway inflammation (left panel) and PAS staining to quantify mucus-producing goblet cells (right panel) in WT and Ptprj^TM–/TM– mice. (D) Total serum IgE levels (relative units) measured by ELISA in WT and Ptprj^TM–/TM– mice in the HDM model. Data for all panels include at least 10 animals per group. Data show the mean ± SEM. (B–D) *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test.

Figure 5. Deletion of CD148 on hematopoietic and endothelial cells does not attenuate AHR.

(A) Flow cytometry plot showing deletion efficiency of CD148 gated on B220⁺ B cells of WT, WT Vav-Cre, Ptprj^TM–/TM–, and Ptprj^{TM–fl/TM–};Vav-Cre mice. Mice of the indicated genotypes were immunized and intranasally challenged with OVA or saline (B–E). (B) Pulmonary resistance measurements after intravenous administration of increasing doses of ACh in control (WT), Ptprj^TM+/TM–, Ptprj^{TM–fl/TM–}, and Ptprj^{TM–fl/TM–};Vav-Cre mice of the C57BL/6 strain. (C) BAL cell counts of total cells, macrophages, eosinophils, lymphocytes, and neutrophils of WT Vav-Cre, Ptprj^{TM–fl/TM–}, and Ptprj^{TM–fl/TM–};Vav-Cre mice. (D) Histologic scoring by a blinded observer of H&E staining (left panel) and PAS staining of lung sections (right panel) in WT Vav-Cre, Ptprj^{TM–fl/TM–}, and Ptprj^{TM–fl/TM–};Vav-Cre mice. (E) Relative OVA-specific serum IgE levels measured by ELISA in WT Vav-Cre, Ptprj^{TM–fl/TM–}, and Ptprj^{TM–fl/TM–};Vav-Cre mice. Data for all panels show the means ± SEM, with 8 to 15 animals per group. *P < 0.05, 2-tailed Student’s t test.

Figure 6. Partial protection from AHR in mice with deletion of CD148 from smooth muscle cells.

(A) Immunofluorescence staining of mouse tracheal smooth muscle with primary hamster antibody to CD148, secondary goat anti-hamster Alexa 488 (green), and α-SMA (SMA, red) as indicated. Right column shows quantitative ImageJ analysis of line scans (areas indicated by white boxes). Mice of the indicated genotypes were immunized and intranasally challenged with OVA or saline (B–E). (B) Pulmonary resistance measurements after intravenous administration of increasing doses of ACh in Ptprj^{TM–fl/TM–fl};TetO-Cre and Ptprj^{TM–fl/TM–fl};SMA-rTTA;TetO-Cre mice of the C57BL/6 strain. *P < 0.05 for Ptprj^{TM–fl/TM–fl};TetO-Cre versus Ptprj^{TM–fl/TM–fl};SMA-rTTA;TetO-Cre at the highest dose of ACh, 2-way ANOVA. (C) BAL cell counts of total cells, macrophages, eosinophils, lymphocytes, and neutrophils of Ptprj^{TM–fl/TM–fl};TetO-Cre and Ptprj^{TM–fl/TM–fl};SMA-rTTA;TetO-Cre mice. (D) Histologic scoring by a blinded observer of H&E-stained sections for degree of inflammation around airways (left panel) and PAS staining for PAS-positive mucus-producing goblet cells (right panel) in WT, Ptprj^{TM–fl/TM–fl};TetO-Cre and Ptprj^{TM–fl/TM–fl};SMA-rTTA;TetO-Cre mice. (E) Relative OVA-specific serum IgE levels measured by ELISA in Ptprj^{TM–fl/TM–fl};TetO-Cre and Ptprj^{TM–fl/TM–fl};SMA-rTTA;TetO-Cre mice. Data for all panels show the mean ± SEM with 8 to 15 animals per group. Scale bar: 50 μm (A). ***P < 0.001, unpaired 2-tailed Student’s t test (D).

Figure 7. CD148-deficient tracheas show diminished contractility, decreased calcium oscillation frequency, and dysregulated SFK signaling in ASM.

Mouse tracheal rings were isolated and stimulated with MCh (A), and contractile force measurements were made with a force transducer. Data are the mean ± SEM with at least 6 tracheal rings per group. *P < 0.05, ***P < 0.001, 2-way ANOVA. (B) Tracheal smooth muscle samples directly isolated from mice were lysed, ultracentrifuged, separated by SDS-PAGE, and transferred to a membrane. Membranes were probed with antibodies against phosphorylated SRC Y527 (inhibitory tyrosine) and total SRC as a loading control. Data shown in this figure are representative of 4 independent experiments. (C) Densitometry quantification of Western blots showing relative intensity of pSRC Y527 compared with total SRC. **P < 0.01, unpaired 2-tailed Student’s t test. (D) Representative calcium oscillations induced by 50 μM MCh in lung slices from BALB/c WT control (upper panel) and Ptprj^TM–/TM– (lower panel) mice. Final fluorescence values are expressed as ratios (F/F₀), normalized to the fluorescence immediately prior to the addition of an agonist (F₀). (E) Average frequency of calcium oscillations, comparing WT control with Ptprj^TM–/TM– mice. Data include 6 animals per group. Data show the mean ± SD. ***P < 0.001, unpaired 2-tailed Student’s t test.

Figure 8. Inhibition of SRC family kinases recapitulates CD148 deficiency and leads to impaired contractility of mouse tracheal rings and human bronchial rings.

Mouse tracheal rings were isolated and pretreated with the specific SRC family kinase inhibitor SU6656 at 10 μM for 12 hours prior to stimulation with MCh (A), and contractile force measurements were made with a force transducer. Mouse tracheal rings from WT (B) or Ptprj^TM–/TM– (C) mice were isolated and pretreated with the specific SRC family kinase inhibitor AZD0530 at 5 μM for 12 hours prior to stimulation with MCh. *P < 0.05, 2-way ANOVA. (D) Human bronchial rings isolated from an explanted human lung were pretreated with SU6656 at 10 μM or vehicle for 1 hour prior to stimulation with MCh and contractile force was measured. Data are the means ± SEM for 5 to 6 mouse tracheal rings (A–C) and 6 human bronchial rings (D) per condition. **P < 0.01, ***P < 0.001, 2-way ANOVA.