Retinoic acid signaling is essential for airway smooth muscle homeostasis
Felicia Chen, … , Ramaswamy Krishnan, Alan Fine
Felicia Chen, … , Ramaswamy Krishnan, Alan Fine
Published August 23, 2018
Citation Information: JCI Insight. 2018;3(16):e120398. https://doi.org/10.1172/jci.insight.120398.
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Research Article Pulmonology

Retinoic acid signaling is essential for airway smooth muscle homeostasis

Abstract

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-β signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-β activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.

Authors

Felicia Chen, Fengzhi Shao, Anne Hinds, Sean Yao, Sumati Ram-Mohan, Timothy A. Norman, Ramaswamy Krishnan, Alan Fine

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Figure 3

RA deficiency in mice results in ASM hypertrophy.

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RA deficiency in mice results in ASM hypertrophy. (A) Forward scatter si...
(A) Forward scatter signals (FSS) of mASM showing higher FSS in RA-deficient mASM (VAD and BMS) compared with RA-sufficient mASM (VAS and CTR, respectively), suggesting larger mASM cell size in RA-deficient lungs (n = 3). (B) Expression of smooth muscle markers Acta2 and Myh11 is increased in VAD and BMS mASM cells compared with their controls (VAS and CTR, respectively), further supporting ASM hypertrophy in RA-deficient mASM (n = 6 per group). (C) Size of hASM cells is increased when cultured in RA-deficient conditions (BMS and DEAB) compared with those cultured in RA-sufficient conditions (CTR) (n = 3). (D) Expression of smooth muscle α-SMA and SM-22α is significantly higher in BMS- and DEAB-treated hASM compared with CTR-treated hASM, indicating hASM hypertrophy when RA signaling is inhibited (n = 3). (E) Representative Western blot of data displayed in D (n = 3; blots shown are from donor 1). (F) Intrinsic cellular contraction of RA-deficient hASM (BMS, DEAB) is higher compared with RA-sufficient hASM (CTR), indicating greater hASM contractility when cultured in RA-deficient conditions (n = 3). (G and H) Immunostaining of Ki-67, a marker of cell proliferation, and α-SMA showing no Ki-67 signal in CTR ASM (G) or BMS ASM (H) in mouse (n = 3). (I and J) Immunostaining of α-SMA and smooth muscle lineage (GFP) in tamoxifen-injected Myh11-CreERT2;mT/mG mice showing membranous GFP signal in all α-SMA+ cells in both CTR and BMS airways, arguing against non-ASM contribution (n = 3). Data in A and C are presented as box-and-whisker plot (see Statistics in Methods section for description). Data in B, D, and F represent the mean ± SEM. Student’s t test was used to calculate P values in A and B (*P < 0.05). Two-way ANOVA was used for statistical analysis in C, D, and F, where Bonferroni’s correction was applied to adjust P values for multiple comparisons (means with different letters are significantly different, P < 0.05). Scale bars: 10 μm (D and F).