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Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model
Journal of Pediatric Surgery (2012) 47, 336–340
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Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model Elke Maria Ruttenstock a,b , Takashi Doi a,c , Jens Dingemann a , Prem Puri a,c,⁎ a
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland Department of Pediatric and Adolescent Surgery, Medical University of Graz, Austria c School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland b
Received 5 November 2011; accepted 10 November 2011
Key words: Retinoic acid; Nitrofen; Pulmonary hypoplasia; Connexin 43; Congenital diaphragmatic hernia
Abstract Purpose: Connexin 43 (Cx43), a major gap junction protein, is necessary for alveologenesis and plays an important role in the differentiation of type II to type I alveolar epithelial cells. Knockout mice of Cx43 display severe pulmonary hypoplasia (PH). Prenatal administration of retinoic acid (RA) is known to stimulate alveologenesis in nitrofen-induced PH. Recent studies revealed that retinoids upregulate Cx43 expression. We hypothesized that gene expression of Cx43 is downregulated during alveologenesis and that administration of RA upregulates Cx43 expression in the nitrofen-induced PH. Methods: Pregnant rats were exposed to olive oil or nitrofen on day 9 (D9) of gestation. Retinoic acid was given intraperitoneally on D18, D19, and D20. Fetal lungs were harvested on D18 and D21 and divided into control, nitrofen, control+RA (D21), and nitrofen+RA (D21). The Cx43 expression levels were determined using reverse transcription polymerase chain reaction and immunohistochemistry. Results: On D18 and D21, Cx43 relative messenger RNA expression levels were significantly downregulated in nitrofen compared with those in the control group. On D21, expression levels of Cx43 were significantly upregulated in nitrofen+RA and control+RA compared with those in nitrofen group. Immunohistochemical studies confirmed these results. Conclusion: Downregulation of Cx43 expression may interfere with normal alveologenesis. Upregulation of Cx43 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in nitrofen-induced PH. © 2012 Elsevier Inc. All rights reserved.
Presented at the 58th Annual Meeting of the British Association of Paediatric Surgeons, Belfast, Northern Ireland, July 20-22, 2011. ⁎ Corresponding author. The National Children's Research Centre, Our Lady's Children's Hospital, Dublin 12, Ireland. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2011.11.026
In infants with congenital diaphragmatic hernia (CDH) pulmonary hypoplasia (PH) is considered to be one of the principle contributors to the high mortality [1-4]. Although the exact mechanism of PH in CDH is still not completely understood, there is increasing evidence that retinoids play an important role in its pathogenesis [5].
Cx43 gene expression in pulmonary hypoplasia The nitrofen-induced CDH in the rodent model is a wellestablished experimental model, reproducing Bochdalek type of diaphragmatic defects and associated PH similar to the human situation [6-8]. Although studies from our laboratory have recently shown that several signalling pathways are altered in the nitrofen-induced hypoplastic lung, the precise mechanisms by which nitrofen acts are still unclear [9-11]. Retinoids, a group of molecules derived from vitamin A, are essential for normal development of various organs including fetal lung morphogenesis [12]. Retinoic acid (RA), the active metabolite of vitamin A, is known to be in particular crucial for each developmental stages of the lung [13]. Recent studies have suggested that the retinoid signalling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung [14,15]. Previous work from our laboratory has revealed that RA rescues PH in the nitrofen-induced hypoplastic fetal rat lung explants [16]. It has also been reported that prenatal treatment with RA promotes alveologenesis by increasing the number of alveoli and gas exchange surface area [17,18]. In addition, it has further been reported that prenatal RA upregulates the expression levels of several genes that are involved in RSP in the nitrofen-induced hypoplastic lung [18]. However, the exact molecular mechanisms by which RA acts in the nitrofen-induced PH is still largely unknown. Connexin 43 (Cx43), a major gap junction protein [19], is necessary for normal alveologenesis and plays an important role in the differentiation of type II alveolar epithelial cells (AECsII) into AECsI [20]. It has been shown that lungs of Cx43 knockout mice display features of severe PH, which are also seen in the nitrofen-induced PH [20]. Furthermore, in vitro studies have demonstrated that retinoids increase Cx43 expression in rodents as well as in humans [21-24]. We designed this study to test the hypothesis that gene expression levels of Cx43 are downregulated during the later stages of lung development and that prenatal administration of RA upregulates Cx43 expression in the nitrofen-induced PH.
337 control+RA (n = 8), nitrofen (n = 16), and nitrofen+RA (n = 16) on D21. To obtain representative numbers, the fetuses in each group came from at least 4 different dams. The Department of Health and Children approved the protocol of these animal experiments (reference B100/4142) under the Cruelty to Animals Act, 1876, as amended by European Communities Regulations 2002 and 2005.
1.2. Isolation of messenger RNA and real-time reverse transcription polymerase chain reaction The peripheral region of the left lung of each fetus was suspended in TRIzol Reagent (Invitrogen, Carlsbad, Calif) immediately after dissection, quick frozen in liquid nitrogen, and stored at −20°C. After thawing frozen samples, they were homogenized with a pellet pestle (Daigger, Vernon Hills, Ill) and total messenger RNA (mRNA) of the lung tissue was isolated according to recommended protocol (Invitrogen). Total mRNA quantification was performed spectrophotometrically (ND-1000 UV-Vis Spectrophotometer, NanoDrop, Wilmington, Del). Synthesis of complementary DNA was performed using Transcript High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's protocol. After reverse transcription was carried out according to previously published protocols [22], real-time polymerase chain reaction was performed using LightCycler 480SYBR Green I Master (Roche Diagnostics) according to the manufacturer's protocol. The specific primer set used in this study is listed in supplemental Table 1, and 45 cycles of amplification were carried out as previously described [24]. Relative levels of gene expression were measured using Light Cycler 480 (Roche Diagnostics) according to the manufacturer's instruction. The mRNA expression levels of Cx43 were normalized to β-actin mRNA expression levels in each sample.
1.3. Immunohistochemistry
1. Materials and methods 1.1. Animals and drugs Adult Sprague-Dawley rats were mated, and the presence of spermatozoids in the vaginal smear was considered as proof of pregnancy, and the day of observation was determined gestational day 0 (D0). Pregnant rats were then randomly divided into 2 groups. On D9 (term, 22 days), the rats in the experimental group received 100 mg of nitrofen (WAKO Chemicals, Osaka, Japan) dissolved in 1 ml of olive oil intragastrically under short anaesthesia, whereas those in control received only vehicle. Five milligram per kilogram of RA (Sigma-Aldrich, Baden-Wuerttemberg, Germany) was given intraperitoneally under short anaesthesia on D18, D19, and D20. Fetuses were harvested by caesarean delivery on D18 and D21. Fetal lungs were divided into 2 groups: control (n = 8) and nitrofen (n = 16) on D18 and 4 groups: control (n = 8),
The paraffin-embedded left lungs were sectioned at a thickness of 5 μm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled water. Immunohistochemistry was performed according to a previously published protocol from our laboratory [14] using Table 1 Primers for quantitative real-time reverse transcription polymerase chain reaction Gene β-actin Forward Reverse Cx43 Forward Reverse
Sequence (5′-3′)
Product size (base pair)
TTG GAT GCC TGT GGT CTG TC 108 TAG AGC CAC CAA TCC ACA CA TGG GTT TTG AGG AGA GGA GA 104 CTT GGA CCT TGT CCA GAA GC
338
E.M. Ruttenstock et al.
a primary antibody specific for Cx43 (LOT sc-59949; 1:100, Santa Cruz Biotechnology, Santa Cruz, Calif) diluted in phosphate buffered saline, and incubated overnight at 4°C. Examination of the fetal lungs under light microscopy was performed independently by 3 investigators.
1.4. Statistical analysis All numerical data are presented as means ± standard deviation. To test the normality distribution of the groups, Kolmogorov-Smirnov test was used. Differences between groups at D18 and D21 were tested by using Tukey-Kramer post hoc test following 1-way analysis of variance or Scheffe's F test following Kruskal-Wallis test. KolmogorovSmirnov test for normality was used in determining use of Kruskal-Wallis test. Statistical significance was accepted at P values of less than .05.
2. Results 2.1. Relative messenger RNA expression levels of Cx43 in fetal rat lungs on D18 and D21 On D18, the relative mRNA expression levels of pulmonary Cx43 were significantly decreased in the nitrofen group compared with those in the control group (P b .05) (Table 2). On D21, the expression levels of Cx43 gene were significantly downregulated in nitrofen group compared with those in the control group. After prenatal treatment with RA, pulmonary gene expression levels of Cx43 were significantly upregulated in control+RA and nitrofen+RA group compared with those in the nitrofen group (P b .05) (Table 2).
2.2. Protein expression of Cx43 in fetal rat lungs on D18 and D21 To determine whether the quantitatively increased amounts of Cx43 transcripts were reflected in the qualitative Table 2
Relative mRNA expression levels of Cx43
Control Nitrofen Control+RA Nitrofen+RA
D18
D21
9.99 (±6.51) 3.91 (±2.49) ⁎
22.47 (±5.49) 3.05 (±2.73) ⁎ 17.90 (±4.25) ⁎⁎ 14.26 (±2.60) ⁎⁎
The relative mRNA expression levels of Cx43 in the fetal lung on D18 (control, n = 8; nitrofen, n = 16) and on D21 (control, n = 8; control+RA, n = 8; nitrofen, n = 16; nitrofen+RA, n = 16). The expression levels of Cx43 are significantly downregulated in nitrofen group compared with those in controls on D18 (⁎P b .05). On D21, Cx43 gene expression levels are significantly increased in nitrofen+RA and control+RA compared with those in nitrofen group (⁎⁎P b .01). ⁎ P b .05 vs control. ⁎⁎ P b .01 vs nitrofen.
amount of the proteins themselves in the nitrofen-induced hypoplastic lung, immunohistochemical studies were performed on D18 and D21 (Fig. 1). Cx43 protein expression was localized in the proximal alveolar epithelium of control lungs. Immunoreactivity of Cx43 was markedly diminished in nitrofen lungs compared with that in control lungs on D18 as well as on D21. After prenatal administration of RA, immunoreactivity of Cx43 was markedly increased in control+RA and nitrofen+RA lungs compared with that in CDH lungs on D21 (Fig. 1).
3. Discussion In patients with CDH, PH still remains one of the major determinants of the high morbidity and the mortality [4]. In addition to a reduction in bronchial divisions in the early lung maturation, arrest in alveolar development during late gestation is observed in patients with CDH. In these patients, alveologenesis in late lung morphogenesis has been shown to be disrupted with thickened intraalveolar septa and diminished air spaces [25]. In fetal development, RSP plays an important role by binding the RA, the active metabolite of vitamin A, to retinoic acid receptors (RARs) and retinoid X receptors [13]. Data from several animal studies have linked perturbations of RSP to CDH. Vitamin A-deficient mice and RARs double knockouts have shown severe phenotype of PH [26]. Administration of exogenous RA has been reported to induce alveolar regeneration in which alveolar septation has been blocked [27]. In the nitrofen model of CDH, the RSP has been known to be disrupted [28]. Several studies from our laboratory have demonstrated that prenatal treatment with RA in the late gestation upregulates the expression levels of several genes that are associated with RSP and rescuing nitrofen-induced PH [4,12-14]. Although only limited literatures have reported the relationship between retinol levels and CDH in humans to date, a clinical study demonstrated that there was a 50% reduction in plasma retinol and retinol-binding protein levels in a group of 11 CDH in newborn compared with those in matching controls [29]. Another study has recently showed significantly lower retinol and retinol-binding protein levels in newborns with CDH compared with those in control newborns [30]. These clinical data along with the extensive experimental evidence in the animal model of CDH suggests that RA may have therapeutic potential in reverting lung hypoplasia in CDH. Connexin 43 is a major gap junction protein. Gap junctions contribute not only to cellular homeostasis such as ion exchange and volume control but also to cellular proliferation and differentiation in various type of organ morphogenesis [19,20]. In fetal lung development, functional analysis of gap junctions revealed that Cx43 is expressed between AECs [31]. Connexin 43 is known to be
Cx43 gene expression in pulmonary hypoplasia
339
Fig. 1 Connexin 43 immunohistochemistry on D18 and D21. On D18, Cx43 is predominantly expressed in the proximal alveolar epithelium in control lungs. Diminished Cx43 protein expression is observed in nitrofen lungs. On D21, Cx43 is predominantly expressed in control lungs compared with that in nitrofen lungs. Connexin 43 also shows markedly increased immunoreactivity in control+RA and nitrofen+RA lungs compared with that in nitrofen lungs.
fundamental for alveologenesis, playing an important role in the differentiation of AECsII to AECsI [20]. The Cx43 knockout mice have shown severe PH with disruption of alveologenesis, resulting in death at birth because of a failure in pulmonary gas exchange [20]. Furthermore, Cx43 is a downstream factor of steroid-thyroid-retinoid nuclear receptor superfamily. It has been recently revealed that molecular signalling involved in this superfamily is disrupted in murine hypoplastic lungs leading to reduced number of alveoli and abnormal alveolar structure, which may be attributed to downregulation of RARs [32]. Furthermore, it has been recently reported that either gain or loss of Cx43 function is involved in the pathogenesis of cardiac malformations in experimental CDH and that Vitamin A administration can rescue them [19,23,33]. In the present study, we clearly showed that Cx43 gene expression was significantly downregulated in the nitrofeninduced hypoplastic lung compared with that in controls both on D18 and on D21, suggesting that downregulation of Cx43 may contribute to PH in the nitrofen CDH rat model. In addition, we showed that pulmonary Cx43 gene expression was significantly upregulated after prenatal treatment with RA in the nitrofen CDH model during the late stage of lung development. Immunohistochemical studies validated these real-time polymerase chain reaction results by showing markedly increased immunoreactivity of Cx43 in the alveolar epithelium of nitrofen+RA lung compared with that in nitrofen-induced hypoplastic lung. Taken together, present findings suggest that RA may promote lung growth
and maturation in the nitrofen-induced PH by stimulating Cx43-mediated alveologenesis.
References [1] Gaxiola A, Varon J, Valladolid G. Congenital diaphragmatic hernia: an overview of the etiology and current management. Acta Paediatr 2009;98:621-7. [2] Keijzer R, Puri P. Congenital diaphragmatic hernia. Semin Pediatr Surg 2010;19:180-5. [3] Stege G, Fenton A, Jaffray B. Nihilism in the 1990s: the true mortality of congenital diaphragmatic hernia. Pediatrics 2003;112:532-5. [4] Gosche JR, Islam S, Boulanger SC. Congenital diaphragmatic hernia: searching for answers. Am J Surg 2005;190:324-32. [5] Gallot D, Marceau G, Coste K, et al. Congenital diaphragmatic hernia: a retinoid-signaling pathway disruption during lung development? Birth Defects Res A Clin Mol Teratol 2005;73:523-31. [6] Noble BR, Babiuk RP, Clugston RD, et al. Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 2007;293:1079-87. [7] Kluth D, Kangah R, Reich P, et al. Nitrofen-induced diaphragmatic hernias in rats: an animal model. J Pediatr Surg 1990;25:850-4. [8] Tenbrick R, Tibboel D, Gaillard JL, et al. Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 1990;25:426-9. [9] Doi T, Ruttenstock E, Dingemann J, et al. Spatiotemporal alteration in phosphatidylinositide 3-kinase-serine/threonine protein kinase B signaling in the nitrofen-induced hypoplastic lung. J Pediatr Surg 2010;45:366-71. [10] Ruttenstock E, Doi T, Dingemann J, et al. Insulinlike growth factor receptor type 1 and type 2 are downregulated in the nitrofen-induced hypoplastic lung. J Pediatr Surg 2010;45:1349-53.
340 [11] Ruttenstock E, Doi T, Dingemann J, et al. Insulin receptor is downregulated in the nitrofen-induced hypoplastic lung. J Pediatr Surg 2010;45:948-52. [12] Ross SA, McCaffery PJ, Drager UC, et al. Retinoids in embryonal development. Physiol Rev 2000;80:1021-54. [13] Maden M. Retinoids in lung development and regeneration. Curr Top Dev Biol 2004;61:153-89. [14] Nakazawa N, Takayasu H, Montedonico S, et al. Altered regulation of retinoic acid synthesis in nitrofen-induced hypoplastic lung. Pediatr Surg Int 2007;23:391-6. [15] Thebaud B, Tibboel D, Rambaud C, et al. Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats. Am J Physiol 1999;277:423-9. [16] Montedonico S, Nakazawa N, Puri P. Retinoic acid rescues lung hypoplasia in nitrofen-induced hypoplastic foetal rat lung explants. Pediatr Surg Int 2006;22:2-8. [17] Montedonico S, Sugimoto K, Felle P, et al. Prenatal treatment with retinoic acid promotes pulmonary alveologenesis in the nitrofen model of congenital diaphragmatic hernia. J Pediatr Surg 2008;43:500-7. [18] Doi T, Sugimoto K, Puri P. Prenatal retinoic acid up-regulates pulmonary gene expression of COUP-TFII, FOG2, and GATA4 in pulmonary hypoplasia. J Pediatr Surg 2009;44:1933-7. [19] Le M, Li Z, Cilley RE, et al. Connexin 43 gene expression in mice with cardiopulmonary developmental defects. Front Biosci 2006;11: 3014-25. [20] Nagata K, Masumoto K, Esumi G, et al. Connexin 43 plays an important role in lung development. J Pediatr Surg 2009;44:2296-301. [21] Bex V, Mercier T, Chaumontet C, et al. Retinoic acid enhances connexin 43 expression at the post-transcriptional level in rat liver epithelial cells. Cell Biochem Funct 1995;13:69-77. [22] Tanmahasamut P, Sidell N. Up-regulation of gap junctional intercellular communication and connexin 43 expression by retinoic acid in human endometrial stromal cells. J Clin Endocrinol Metab 2005;90: 4151-6.
E.M. Ruttenstock et al. [23] Gonzalez-Reyes S, Fernandez-Dumont V, Calonge WM, et al. Expression of connexin 43 in the hearts of rat embryos exposed to nitrofen and effects of vitamin A on it. Pediatr Surg Int 2006;22:61-5. [24] Ruttenstock E, Doi T, Dingemann J, et al. Prenatal administration of retinoic acid upregulates insulin-like growth factor receptors in the nitrofen-induced hypoplastic lung. Birth Defects Res B Dev Reprod Toxicol 2011;92:148-51. [25] Suen HC, Bloch KD, Donahoe PK. Antenatal glucocorticoid corrects pulmonary immaturity in experimentally induced congenital diaphragmatic hernia in rats. Pediatr Res 1994;35:523-9. [26] Antipatis C, Ashworth CJ, Grant G, et al. Effects of maternal vitamin A status on fetal heart and lung: changes in expression of key developmental genes. Am J Physiol 1998;275:1184-91. [27] Massaro GD, Massaro D. Retinoic acid treatment partially rescues failed septation in rats and in mice. Am J Physiol Lung Cell Mol Physiol 2000;278:955-60. [28] Nakazawa N, Montedonico S, Takayasu H, et al. Disturbance of retinol transportation causes nitrofen-induced hypoplastic lung. J Pediatr Surg 2007;42:345-9. [29] Major D, Cadenas M, Fournier L, et al. Retinol status of newborn infants with congenital diaphragmatic hernia. Pediatr Surg Int 1998;13: 547-9. [30] Beurskens LW, Tibboel D, Lindemans J, et al. Retinol status of newborn infants is associated with congenital diaphragmatic hernia. Pediatrics 2010;126:712-20. [31] Rannels DE. Gap junction communication in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 2001;280:1083-4. [32] Chinoy MR, Chi X, Cilley RE. Down-regulation of regulatory proteins for differentiation and proliferation in murine fetal hypoplastic lungs: altered mesenchymal-epithelial interactions. Pediatr Pulmonol 2001;32:129-41. [33] Chen P, Xie LJ, Huang GY, et al. Mutations of connexin 43 in fetuses with congenital heart malformations. Chin Med J (Engl) 2005;118: 971-6.
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Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model Elke Maria Ruttenstock a,b , Takashi Doi a,c , Jens Dingemann a , Prem Puri a,c,⁎ a
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland Department of Pediatric and Adolescent Surgery, Medical University of Graz, Austria c School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland b
Received 5 November 2011; accepted 10 November 2011
Key words: Retinoic acid; Nitrofen; Pulmonary hypoplasia; Connexin 43; Congenital diaphragmatic hernia
Abstract Purpose: Connexin 43 (Cx43), a major gap junction protein, is necessary for alveologenesis and plays an important role in the differentiation of type II to type I alveolar epithelial cells. Knockout mice of Cx43 display severe pulmonary hypoplasia (PH). Prenatal administration of retinoic acid (RA) is known to stimulate alveologenesis in nitrofen-induced PH. Recent studies revealed that retinoids upregulate Cx43 expression. We hypothesized that gene expression of Cx43 is downregulated during alveologenesis and that administration of RA upregulates Cx43 expression in the nitrofen-induced PH. Methods: Pregnant rats were exposed to olive oil or nitrofen on day 9 (D9) of gestation. Retinoic acid was given intraperitoneally on D18, D19, and D20. Fetal lungs were harvested on D18 and D21 and divided into control, nitrofen, control+RA (D21), and nitrofen+RA (D21). The Cx43 expression levels were determined using reverse transcription polymerase chain reaction and immunohistochemistry. Results: On D18 and D21, Cx43 relative messenger RNA expression levels were significantly downregulated in nitrofen compared with those in the control group. On D21, expression levels of Cx43 were significantly upregulated in nitrofen+RA and control+RA compared with those in nitrofen group. Immunohistochemical studies confirmed these results. Conclusion: Downregulation of Cx43 expression may interfere with normal alveologenesis. Upregulation of Cx43 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in nitrofen-induced PH. © 2012 Elsevier Inc. All rights reserved.
Presented at the 58th Annual Meeting of the British Association of Paediatric Surgeons, Belfast, Northern Ireland, July 20-22, 2011. ⁎ Corresponding author. The National Children's Research Centre, Our Lady's Children's Hospital, Dublin 12, Ireland. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2011.11.026
In infants with congenital diaphragmatic hernia (CDH) pulmonary hypoplasia (PH) is considered to be one of the principle contributors to the high mortality [1-4]. Although the exact mechanism of PH in CDH is still not completely understood, there is increasing evidence that retinoids play an important role in its pathogenesis [5].
Cx43 gene expression in pulmonary hypoplasia The nitrofen-induced CDH in the rodent model is a wellestablished experimental model, reproducing Bochdalek type of diaphragmatic defects and associated PH similar to the human situation [6-8]. Although studies from our laboratory have recently shown that several signalling pathways are altered in the nitrofen-induced hypoplastic lung, the precise mechanisms by which nitrofen acts are still unclear [9-11]. Retinoids, a group of molecules derived from vitamin A, are essential for normal development of various organs including fetal lung morphogenesis [12]. Retinoic acid (RA), the active metabolite of vitamin A, is known to be in particular crucial for each developmental stages of the lung [13]. Recent studies have suggested that the retinoid signalling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung [14,15]. Previous work from our laboratory has revealed that RA rescues PH in the nitrofen-induced hypoplastic fetal rat lung explants [16]. It has also been reported that prenatal treatment with RA promotes alveologenesis by increasing the number of alveoli and gas exchange surface area [17,18]. In addition, it has further been reported that prenatal RA upregulates the expression levels of several genes that are involved in RSP in the nitrofen-induced hypoplastic lung [18]. However, the exact molecular mechanisms by which RA acts in the nitrofen-induced PH is still largely unknown. Connexin 43 (Cx43), a major gap junction protein [19], is necessary for normal alveologenesis and plays an important role in the differentiation of type II alveolar epithelial cells (AECsII) into AECsI [20]. It has been shown that lungs of Cx43 knockout mice display features of severe PH, which are also seen in the nitrofen-induced PH [20]. Furthermore, in vitro studies have demonstrated that retinoids increase Cx43 expression in rodents as well as in humans [21-24]. We designed this study to test the hypothesis that gene expression levels of Cx43 are downregulated during the later stages of lung development and that prenatal administration of RA upregulates Cx43 expression in the nitrofen-induced PH.
337 control+RA (n = 8), nitrofen (n = 16), and nitrofen+RA (n = 16) on D21. To obtain representative numbers, the fetuses in each group came from at least 4 different dams. The Department of Health and Children approved the protocol of these animal experiments (reference B100/4142) under the Cruelty to Animals Act, 1876, as amended by European Communities Regulations 2002 and 2005.
1.2. Isolation of messenger RNA and real-time reverse transcription polymerase chain reaction The peripheral region of the left lung of each fetus was suspended in TRIzol Reagent (Invitrogen, Carlsbad, Calif) immediately after dissection, quick frozen in liquid nitrogen, and stored at −20°C. After thawing frozen samples, they were homogenized with a pellet pestle (Daigger, Vernon Hills, Ill) and total messenger RNA (mRNA) of the lung tissue was isolated according to recommended protocol (Invitrogen). Total mRNA quantification was performed spectrophotometrically (ND-1000 UV-Vis Spectrophotometer, NanoDrop, Wilmington, Del). Synthesis of complementary DNA was performed using Transcript High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's protocol. After reverse transcription was carried out according to previously published protocols [22], real-time polymerase chain reaction was performed using LightCycler 480SYBR Green I Master (Roche Diagnostics) according to the manufacturer's protocol. The specific primer set used in this study is listed in supplemental Table 1, and 45 cycles of amplification were carried out as previously described [24]. Relative levels of gene expression were measured using Light Cycler 480 (Roche Diagnostics) according to the manufacturer's instruction. The mRNA expression levels of Cx43 were normalized to β-actin mRNA expression levels in each sample.
1.3. Immunohistochemistry
1. Materials and methods 1.1. Animals and drugs Adult Sprague-Dawley rats were mated, and the presence of spermatozoids in the vaginal smear was considered as proof of pregnancy, and the day of observation was determined gestational day 0 (D0). Pregnant rats were then randomly divided into 2 groups. On D9 (term, 22 days), the rats in the experimental group received 100 mg of nitrofen (WAKO Chemicals, Osaka, Japan) dissolved in 1 ml of olive oil intragastrically under short anaesthesia, whereas those in control received only vehicle. Five milligram per kilogram of RA (Sigma-Aldrich, Baden-Wuerttemberg, Germany) was given intraperitoneally under short anaesthesia on D18, D19, and D20. Fetuses were harvested by caesarean delivery on D18 and D21. Fetal lungs were divided into 2 groups: control (n = 8) and nitrofen (n = 16) on D18 and 4 groups: control (n = 8),
The paraffin-embedded left lungs were sectioned at a thickness of 5 μm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled water. Immunohistochemistry was performed according to a previously published protocol from our laboratory [14] using Table 1 Primers for quantitative real-time reverse transcription polymerase chain reaction Gene β-actin Forward Reverse Cx43 Forward Reverse
Sequence (5′-3′)
Product size (base pair)
TTG GAT GCC TGT GGT CTG TC 108 TAG AGC CAC CAA TCC ACA CA TGG GTT TTG AGG AGA GGA GA 104 CTT GGA CCT TGT CCA GAA GC
338
E.M. Ruttenstock et al.
a primary antibody specific for Cx43 (LOT sc-59949; 1:100, Santa Cruz Biotechnology, Santa Cruz, Calif) diluted in phosphate buffered saline, and incubated overnight at 4°C. Examination of the fetal lungs under light microscopy was performed independently by 3 investigators.
1.4. Statistical analysis All numerical data are presented as means ± standard deviation. To test the normality distribution of the groups, Kolmogorov-Smirnov test was used. Differences between groups at D18 and D21 were tested by using Tukey-Kramer post hoc test following 1-way analysis of variance or Scheffe's F test following Kruskal-Wallis test. KolmogorovSmirnov test for normality was used in determining use of Kruskal-Wallis test. Statistical significance was accepted at P values of less than .05.
2. Results 2.1. Relative messenger RNA expression levels of Cx43 in fetal rat lungs on D18 and D21 On D18, the relative mRNA expression levels of pulmonary Cx43 were significantly decreased in the nitrofen group compared with those in the control group (P b .05) (Table 2). On D21, the expression levels of Cx43 gene were significantly downregulated in nitrofen group compared with those in the control group. After prenatal treatment with RA, pulmonary gene expression levels of Cx43 were significantly upregulated in control+RA and nitrofen+RA group compared with those in the nitrofen group (P b .05) (Table 2).
2.2. Protein expression of Cx43 in fetal rat lungs on D18 and D21 To determine whether the quantitatively increased amounts of Cx43 transcripts were reflected in the qualitative Table 2
Relative mRNA expression levels of Cx43
Control Nitrofen Control+RA Nitrofen+RA
D18
D21
9.99 (±6.51) 3.91 (±2.49) ⁎
22.47 (±5.49) 3.05 (±2.73) ⁎ 17.90 (±4.25) ⁎⁎ 14.26 (±2.60) ⁎⁎
The relative mRNA expression levels of Cx43 in the fetal lung on D18 (control, n = 8; nitrofen, n = 16) and on D21 (control, n = 8; control+RA, n = 8; nitrofen, n = 16; nitrofen+RA, n = 16). The expression levels of Cx43 are significantly downregulated in nitrofen group compared with those in controls on D18 (⁎P b .05). On D21, Cx43 gene expression levels are significantly increased in nitrofen+RA and control+RA compared with those in nitrofen group (⁎⁎P b .01). ⁎ P b .05 vs control. ⁎⁎ P b .01 vs nitrofen.
amount of the proteins themselves in the nitrofen-induced hypoplastic lung, immunohistochemical studies were performed on D18 and D21 (Fig. 1). Cx43 protein expression was localized in the proximal alveolar epithelium of control lungs. Immunoreactivity of Cx43 was markedly diminished in nitrofen lungs compared with that in control lungs on D18 as well as on D21. After prenatal administration of RA, immunoreactivity of Cx43 was markedly increased in control+RA and nitrofen+RA lungs compared with that in CDH lungs on D21 (Fig. 1).
3. Discussion In patients with CDH, PH still remains one of the major determinants of the high morbidity and the mortality [4]. In addition to a reduction in bronchial divisions in the early lung maturation, arrest in alveolar development during late gestation is observed in patients with CDH. In these patients, alveologenesis in late lung morphogenesis has been shown to be disrupted with thickened intraalveolar septa and diminished air spaces [25]. In fetal development, RSP plays an important role by binding the RA, the active metabolite of vitamin A, to retinoic acid receptors (RARs) and retinoid X receptors [13]. Data from several animal studies have linked perturbations of RSP to CDH. Vitamin A-deficient mice and RARs double knockouts have shown severe phenotype of PH [26]. Administration of exogenous RA has been reported to induce alveolar regeneration in which alveolar septation has been blocked [27]. In the nitrofen model of CDH, the RSP has been known to be disrupted [28]. Several studies from our laboratory have demonstrated that prenatal treatment with RA in the late gestation upregulates the expression levels of several genes that are associated with RSP and rescuing nitrofen-induced PH [4,12-14]. Although only limited literatures have reported the relationship between retinol levels and CDH in humans to date, a clinical study demonstrated that there was a 50% reduction in plasma retinol and retinol-binding protein levels in a group of 11 CDH in newborn compared with those in matching controls [29]. Another study has recently showed significantly lower retinol and retinol-binding protein levels in newborns with CDH compared with those in control newborns [30]. These clinical data along with the extensive experimental evidence in the animal model of CDH suggests that RA may have therapeutic potential in reverting lung hypoplasia in CDH. Connexin 43 is a major gap junction protein. Gap junctions contribute not only to cellular homeostasis such as ion exchange and volume control but also to cellular proliferation and differentiation in various type of organ morphogenesis [19,20]. In fetal lung development, functional analysis of gap junctions revealed that Cx43 is expressed between AECs [31]. Connexin 43 is known to be
Cx43 gene expression in pulmonary hypoplasia
339
Fig. 1 Connexin 43 immunohistochemistry on D18 and D21. On D18, Cx43 is predominantly expressed in the proximal alveolar epithelium in control lungs. Diminished Cx43 protein expression is observed in nitrofen lungs. On D21, Cx43 is predominantly expressed in control lungs compared with that in nitrofen lungs. Connexin 43 also shows markedly increased immunoreactivity in control+RA and nitrofen+RA lungs compared with that in nitrofen lungs.
fundamental for alveologenesis, playing an important role in the differentiation of AECsII to AECsI [20]. The Cx43 knockout mice have shown severe PH with disruption of alveologenesis, resulting in death at birth because of a failure in pulmonary gas exchange [20]. Furthermore, Cx43 is a downstream factor of steroid-thyroid-retinoid nuclear receptor superfamily. It has been recently revealed that molecular signalling involved in this superfamily is disrupted in murine hypoplastic lungs leading to reduced number of alveoli and abnormal alveolar structure, which may be attributed to downregulation of RARs [32]. Furthermore, it has been recently reported that either gain or loss of Cx43 function is involved in the pathogenesis of cardiac malformations in experimental CDH and that Vitamin A administration can rescue them [19,23,33]. In the present study, we clearly showed that Cx43 gene expression was significantly downregulated in the nitrofeninduced hypoplastic lung compared with that in controls both on D18 and on D21, suggesting that downregulation of Cx43 may contribute to PH in the nitrofen CDH rat model. In addition, we showed that pulmonary Cx43 gene expression was significantly upregulated after prenatal treatment with RA in the nitrofen CDH model during the late stage of lung development. Immunohistochemical studies validated these real-time polymerase chain reaction results by showing markedly increased immunoreactivity of Cx43 in the alveolar epithelium of nitrofen+RA lung compared with that in nitrofen-induced hypoplastic lung. Taken together, present findings suggest that RA may promote lung growth
and maturation in the nitrofen-induced PH by stimulating Cx43-mediated alveologenesis.
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