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. 2012 Jun 1;13(1):42.
doi: 10.1186/1465-9921-13-42.

Maternal smoking and the retinoid pathway in the developing lung

Sara E Manoli  1 , Lacey A Smith, Carrie A Vyhlidal, Chang Hyeok An, Yolanda Porrata, Wellington V Cardoso, Rebecca M Baron, Kathleen J Haley
Affiliations

Maternal smoking and the retinoid pathway in the developing lung

Sara E Manoli et al. Respir Res. .

Abstract

Background: Maternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model.

Methods: Female C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro.

Results: Compared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3-5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells.

Conclusions: A murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.

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Figures

Figure 1
Figure 1
Increased airspace size in mice with tobacco toxin (TT) exposure during lung development compared to tobacco-naïve (NS) mice.A. Representative section of lung tissue stained with hematoxylin and eosin (H&E) from postnatal day 14 (P14) NS mouse (Magnification, 20X) showing normal alveolar size. B. H&E stain of representative section of lung tissue from P14 TT mouse (Magnification, 20X) showing enlarged airspace size, consistent with alveolarization defect. C. The mean linear intercept, which reflects average distance between gas exchange elements, is significantly larger in TT mice compared to NS mice. Data comparison performed using Student’s t-test. Abbreviation: N = number of mice in group. Data are mean ± SEM.
Figure 2
Figure 2
Quantitative PCR analysis of RA pathway components at postnatal day 5 (P5) comparing mice with tobacco toxin exposure during lung development (TT, N = 9) to tobacco naïve (NS, N = 5) mice. All results normalized to expression of 18 S. Compared to NS mice, lung tissue samples from mice with TT had decreased expression of retinoic acid receptors (RAR) alpha and beta, retinoid X receptor (RXR) alpha, nuclear receptor family 2, group F, member 2 (Nr2f2) – also known as chicken ovalbumin upstream promoter transcription factor 2 (COUP TF 2), retinaldehyde dehydrogenase 1 (Raldh1), and cytochrome P450 26b1 (Cyp 26b1). Data are presented as mean ± SEM. NS = tobacco naïve. Data comparison performed using Student’s t-test for normally distributed data and Mann–Whitney for non-normally distributed data.
Figure 3
Figure 3
Western blot analyses for retinoid X receptor alpha (RXRalpha), retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RARbeta), and retinoic acid receptor gamma (RARgamma) expression in distal lung tissue from mice with and without tobacco toxin exposure during development (TT) at postnatal age P3, P5, and P7. Representative Western blots for RXRalpha (A), RARalpha (C), RARbeta (E), and RARgamma (G). Panels B.,D,F, and H: Densitometry analyses of Western blots on distal lung tissue from juvenile mice at postnatal ages P3 with (N = 6) and without (N = 6) TT, P5 (N = 4 each with/without TT), and P7 with (N = 6) and without (N = 5) TT. Compared to tobacco-naïve mice, TT pups expressed significantly decreased RXRalpha at each time point (B): at P3, P = 0.0089, at P5 P = 0.0138, and at P7 P = 0.0227. Decreased expression in the TT mice was also seen for RARalpha, although differences between groups were less than those for RXRalpha, with borderline significance observed at P3 (P = 0.0498) and trends for decreased expression at P5 (P = 0.0709) at P5 and P7 (P = 0.0866) (D). In contrast, TT mice only showed significantly decreased RARbeta at P3 (P < 0.001) (F). The expression of RARgamma was similar to that of RARbeta, with significant decreases seen in the TT mice at P3 (P = 0.0042), with a trend toward decrease at P7 (P = 0.0728). Data comparison performed using Student’s t-test. Abbreviations: RXR = retinoid X receptor, RAR = retinoic acid receptor.
Figure 4
Figure 4
Cigarette smoke condensate (CSC) significantly (P < 0.05) decreases retinoic acid-stimulated (1 μM all groups) response element (RARE) activity as detected by a luciferase reporter plasmid in A549 cells. Luciferase activity was normalized to that of the retinoic acid-stimulated cultures without CSC. Data (mean ± SEM) are from 4 independent experiments. Data comparison performed using Student’s t-test.
Figure 5
Figure 5
Cigarette smoke condensate (CSC) decreases retinoic acid response element bindingin vitro.A: Representative electrophoretic mobility shift assay (EMSA) showing decreased binding of nuclear extracts from A549 cells to the DR5 retinoic acid response element after 1 μM retinoic acid and vehicle (lanes 2 – 5) or 0.50% cigarette smoke condensate (CSC, lanes 6–9) treatment followed by second CSC dose of 0.50% 14 h after the first, with extracts harvested at 15 – 120 min. following this second dose. Similar results were also shown in an independent replication. B: Relative densities of A549 cell nuclear extract/DR5 retinoic acid response element complexes (normalized to free probe) following stimulation with 1 μM retinoic acid and either vehicle or 0.5% CSC as outlined for panel A. Data are mean ± SEM from two independent experiments. Abbreviations: N = negative control lane (lane 1) with equivolume extraction buffer substituted for the nuclear extract; numbers indicate time course in minutes, lane numbers for samples indicated below figure, NE = nuclear extract, CSC = cigarette smoke condensate.
Figure 6
Figure 6
Real-time PCR analysis of postnatal day 5 murine lung tissue showing significantly decreased expression of mRNA (normalized for 18 S expression) for genes regulated by retinoic acid that modulate alveolarization and/or postnatal lung function in mice exposed to tobacco toxin exposure during lung development (TT, N = 5) compared to tobacco-naïve mice (NS, N = 9). Abbreviations: SMA = smooth muscle actin, CD31 = platelet endothelial cell adhesion molecule, VEGF = vascular endothelial cell growth factor alpha, TGF-β = transforming growth factor beta, SPB = surfactant apoprotein beta. Data are presented as mean ± SEM. Data comparison performed with Student’s t-test for normally distributed data and Mann–Whitney for non-normally distributed data.
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