BMP signaling is essential in neonatal surfactant production during respiratory adaptation

Abstract

Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of bone morphogenetic protein (BMP) signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 in embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, whereas prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target, Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression and also stimulate the activity of an Nfatc3 promoter that contains a Smad1-binding site. Therefore, our study suggests that the BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing.

Keywords: Bmpr1a; Nfatc3; Smad1; lung development; neonatal respiratory distress.

Fig. 1.

Bone morphogenetic protein (BMP)-Smad1 signaling was upregulated in neonatal lung immediately after birth, accompanied by increased Abca3 expression. A: dynamic profiles of Smad1 phosphorylation and surfactant protein (Sp)/Abca3 expression in neonatal lungs. Lung tissues were harvested from the mice just before delivery (embryonic day 19, E19) and different hours after birth. The related proteins were detected by Western blot, and Gapdh was used as a loading control. B: quantitative analysis of the related protein band intensities by densitometry. Experiments were repeated 3 times, and significant increases of pSmad1 and Abca3 were detected at 4 and 12 h (*P = 0.002, @P = 0.005, #P = 0.007, and $P = 0.004). C: gene expression in lung tissues at the mRNA level was measured by real-time PCR. *P = 0.017, #P = 0.002.

Fig. 2.

Alk3 conditional knockout (CKO) in perinatal mouse lung epithelial cells. A: Alk3 protein in total lung tissue lysate was detected by Western blot. Lung samples were isolated from E18.5-induced Alk3 homozygous CKO, Alk3 heterozygous CKO (HT), or wild-type (WT) mice 4 h after birth. B: Alk3 expression in the neonatal lung (4 h after birth) was stained with anti-Alk3 antibody (green). Cell nuclei were counterstained with DAPI (blue).

Fig. 3.

Neonatal Alk3 CKO mice died from respiratory distress. A: neonatal mice with different Alk3 genotypes. B: altered survival was detected in Alk3 CKO mice compared with the neonates with Alk3 WT and HT genotypes. *P = 0.014. C: hematoxylin and eosin (H and E)-stained tissue sections of the lungs isolated from the prenatal (E19) or postnatal mice (4 h after birth) with the indicated genotypes. D: morphometric analyses of peripheral airspace areas shown in H and E-stained lung tissue sections, measured by percentage of peripheral airspace area [(airspace area in tissue section/tissue section area) × 100]. Comparison was performed between WT and Alk3 HT lungs or WT and Alk3 CKO lungs, *P < 0.01.

Fig. 4.

Reduced expression of Abca3 and surfactant proteins in Alk3 CKO lungs 4 h after birth. A: gene expression at the mRNA level in lung tissue was compared by real-time PCR. *P = 0.002, #P = 0.009, @P = 0.003 compared with WT controls. B: related proteins in total lung tissue lysate were compared by Western blot. C: quantitative analyses of protein bands detected in B using densitometry. *P = 0.031, #P = 0.046, @P = 0.040, $P = 0.047, &P = 0.013 compared with WT controls. D: secreted SP-B and SP-C in equal volume of bronchoalveolar lavage (BAL), measured by ELISA, was compared between WT and Alk3 CKO samples. *P = 0.013, #P = 0.012. E: altered distributions of the related proteins in neonatal lung tissues with Alk3 CKO genotypes were also detected by immunofluorescence staining as indicated in the pictures. Cell nuclei were counterstained with DAPI (blue).

Fig. 5.

Altered expression of the related proteins in isolated lung epithelial cells from neonatal Alk3 CKO lungs vs. those from WT littermate controls. A: Western blot detection. B: quantitative analysis of the related protein band intensities in A by densitometry. Experiments were repeated 3 times. Reduced protein expression of Alk3 (*P < 0.001), Abca3 (*P = 0.01), precursors of SP-B (*P = 0.045) and SP-C (*P = 0.043), and Nfatc3 (*P = 0.004) was verified in Alk3 CKO lungs compared with the WT controls.

Fig. 6.

Neonatal mice with lung epithelium-specific Smad1 CKO induced from E18.5 suffered from atelectasis accompanied by reduced expression of surfactant proteins and Abca3. A: comparison of lung histology between Smad1 CKO and WT littermate control before and after birth [E19 vs. 4 h after birth (P1)]. B: altered expression of surfactant proteins and Abca3 at the mRNA level was compared between neonatal Smad1 CKO lungs and WT littermate controls. *P = 0.002, #P = 0.003, @P = 0.001 (n = 3 for each genotype). C and D: altered protein expression in Smad1 CKO lung tissue was detected by Western blot (C) and quantified by densitometry (D). Experiments were repeated 3 times, and significant reductions of Smad1 (*P = 0.026), Abca3 (*P = 0.024), SP-B (*P = 0.046 and #P = 0.029), and SP-C (*P = 0.013 and #P = 0.019) were detected.

Fig. 7.

Altered expression of the related proteins in isolated lung epithelial cells from neonatal Smad1 CKO lungs vs. those from WT littermate controls. A: Western blot detection. B: quantitative analysis of the related protein band intensities in A by densitometry. Experiments were repeated 3 times. Reduced protein expression of Alk3 (*P < 0.001), Abca3 (*P = 0.001), precursors of SP-B (*P = 0.028) and SP-C (*P = 0.010), and Nfatc3 (*P < 0.001) was verified in Smad1 CKO lungs compared with the WT controls.

Fig. 8.

BMP4-Smad1 signaling directly upregulated Nfatc3 expression. A: chromatin immunoprecipitation combined with microarray technology (ChIP-chip) analysis of E18.5 WT lung tissues suggested that Nfatc3 promoter DNA might interact with Smad1, with peak score 2.5. B: Nfatc3 protein expression in lung tissue, detected by Western blot, was compared between neonatal Alk3 CKO and WT littermates or neonatal Smad1 CKO and WT littermates 4 h after birth. C: quantitative analysis of Nfatc3 protein band intensities detected in B by densitometry. Experiments were repeated 3 times. Significant reduction of Nfatc3 protein expression was verified for Alk3 CKO lungs (*P = 0.026) and Smad1 CKO lungs (*P = 0.046). D and E: in cultured MLE12 cells (D) or primary lung epithelial cells (E), addition of BMP4 (25 ng/ml) significantly stimulated Nfatc3 expression (*P = 0.004 in D, *P = 0.020 in E), as well as expression of Abca3 (*P = 0.049 in D, *P = 0.022 in E), Sftpb (*P = 0.001 in D, *P = 0.007 in E), and Sftpc (*P = 0.017 in D, *P = 0.008 in E) at the mRNA level after 24 h, detected by real-time PCR. Pretreatment of cells with Alk3 inhibitor LDN-193189 (LDN) blocked the above BMP4-stimulated gene expression (Nfatc3: #P = 0.008 in D, #P = 0.013 in E; Abca3: #P = 0.047 in D, #P = 0.008 in E; Sftpb: #P = 0.047 in D, #P = 0.020 in E; Sftpc: #P = 0.028 in D, #P = 0.002 in E). F and G: impacts of BMP4 and/or Alk3 inhibitor LDN on the related protein expression in cultured MLE12 cells (F) and the primary lung epithelial cells (G) were also examined by Western blot.

Fig. 9.

Smad1-binding site on Nfatc3 promoter mediated BMP-Alk3-Smad1 activation of Nfatc3 transcription. A: consensus DNA sequence of Smad1-binding site (bold), which is similar to that reported in mouse Id1 promoter, was found in both human and mouse Nfatc3 promoter by DNA sequence analysis. B: ChIP analysis of E18.5 lung tissue using the indicated antibodies. The coprecipitated Smad1-binding DNA sequence in Nfatc3 promoter was detected by PCR. C: luciferase reporter assay to detect the BMP-Smad1-responsive element in Nfatc3 promoter in MLE12 cells. pNfatc3-luc, a reporter plasmid containing WT Nfatc3 promoter; pNfatc3-mut-luc, a reporter plasmid derived from pNfatc3-luc with a deletion of potential Smad1-binding site; BMP4, 25 ng/ml; LDN, 30 nM. *P < 0.001, #P < 0.001, @P < 0.001. D: activation of BMP signaling by coexpressing CaAlk3 and/or Smad1 significantly enhanced the Nfatc3 reporter activity in MLE12 cells, whereas coexpression with Smurf1, a ubiquitin ligase to degrade Smad1, partially blocked the increase of Nfatc3 reporter activity. *P = 0.001, #P < 0.001, @P < 0.001.