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Maternal smoking during pregnancy decreases Wnt signalling in neonatal mice
  1. M J Blacquière1,
  2. W Timens1,
  3. A van den Berg1,
  4. M Geerlings1,
  5. D S Postma2,
  6. M N Hylkema1
  1. 1Department of Pathology, University Medical Center Groningen, University of Groningen, The Netherlands
  2. 2Department of Pulmonology, University Medical Center Groningen, University of Groningen, The Netherlands
  1. Correspondence to Dr M Hylkema, Department of Pathology, University Medical Center Groningen, Hanzeplein 1, P O Box 196, 9700 RB Groningen, The Netherlands; m.n.hylkema{at}path.umcg.nl

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Epidemiological studies have shown that maternal smoking during pregnancy is a risk factor for the development of asthma. However, the mechanisms underlying the increased risk of developing asthma are largely unknown. We have shown that maternal smoking during pregnancy increases smooth muscle layer and collagen III deposition around the airways in mouse offspring in association with increased airway hyper-responsiveness.1 Other factors also appear to contribute to the development of increased airway hyper-responsiveness. We hypothesise that lung development is such a factor. Since genes in the Wnt-β-catenin pathway are essential for lung development and epithelial stem cell differentiation/expansion, we investigated the effects of maternal smoking during pregnancy on Wnt gene expression in lung tissue from neonatal offspring.

Balb/c mice were exposed to fresh air or cigarette smoke from 3 weeks before conception until delivery. Offspring (n = 16 from non-smoking mothers, n = 18 from smoking mothers) were killed 1 day after birth. RNA was isolated from total lung tissue and qRT-PCR was performed using microfluidic card assay.

We found that maternal smoking during pregnancy decreased the expression of Foxa2, Fzd-7, Egf, Ctnnb1, Fn1 and Pdgfra (encoding forkhead box a2, frizzled receptor 7, epidermal growth factor, β-catenin, fibronectin and platelet-derived growth factor receptor a, respectively) in lung tissue from neonatal offspring (figure 1). Maternal smoking had no effect on the expression levels of Wnt-5 and -7, Fzd-4 and -10, and Pdgfa (encoding wingless proteins 5 and 7, frizzled receptors 4 and 10 and platelet-derived growth factor, respectively) and expression of Dkk1 (encoding dikkopf) was not detected. For an overview of the effects of maternal smoking during pregnancy on the Wnt pathway in the lung tissue of offspring, see figure 2 in the online supplement.

Figure 1

Relative gene expression in lung tissue of offspring from non-smoking mothers (open symbols) and smoking mothers (closed symbols). Data were normalised to GAPDH in order to correct for differences in input using the formula ΔCt = Ct(GAPDH) − Ct(gene of interest). The relative expression levels were calculated by 2−(ΔCt) and levels are given relative to the non-smoking group. Medians from non-smoking mother group  = 1. *p<0.05, **p<0.01.

To our knowledge, this is the first report showing that maternal smoking during pregnancy decreases the expression of several genes involved in Wnt signalling in offspring. We propose that this relates to lung development as follows. Foxa2 decreases alveolarisation and increases goblet cell hyperplasia when conditionally deleted from epithelial cells.2 Interestingly, we have previously shown increased house dust mite-induced goblet cell hyperplasia in offspring from smoking mothers,1 which could have been caused by decreased Foxa2 expression. Furthermore, epidermal growth factor, β-catenin and the downstream Wnt target gene Fn1 were also shown to be involved in branching morphogenesis of the fetal lung.3–5 In addition, Fzd-7 is important in neovessel formation,6 while Pdgfra is involved in both neovessel formation and alveolarisation.7

Together, these findings provide a plausible argument for the effects of maternal smoking during pregnancy on Wnt-β-catenin signalling in the lungs of neonatal offspring. This may contribute to impaired lung development and an increased risk of developing asthma later in life. Moreover, Wnt signalling is involved in many other developmental processes that may thus be affected by maternal smoking during pregnancy. The relationship between defects in Wnt signalling and morphological/functional outcomes in lung tissue from offspring should therefore be investigated in more detail.

References

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Footnotes

  • Funding This work was supported by a grant from the European Commission as part of GABRIEL, a multidisciplinary study to identify the genetic and environmental causes of asthma in the European Community (contract no 018996).

  • Competing interests None.

  • Provenance and Peer review Not commissioned; externally peer reviewed.

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