Down-Regulation of Lung Kruppel-Like Factor in the Nitrofen-Induced Hypoplastic Lung

Abstract

Introduction: Pulmonary hypoplasia is a primary cause of high morbidity and mortality in neonates with Congenital Diaphragmatic Hernia (CDH). However, the precise pathogenesis of PH associated with CDH is still not clearly understood. It has been recently reported that lung Kruppel-like factor (LKLF), a member of the Kruppel-like factor family of transcription factors, is predominantly expressed in lungs and plays an important role in lung morphogenesis and functional maturation. It has been reported that homozygous deletion of LKLF gene in mice results in reduced lung morphogenesis. It is further reported that chimeric mice derived from LKLF^-/- embryonic stem cells exhibit delayed lung development especially in the later gestational stages. We therefore designed this study to test the hypothesis that the LKLF gene is down-regulated during later stages of lung development in nitrofen-induced hypoplastic lungs.

Material and Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups:control, nitrofen without CDH(CDH(−)) and nitrofen with CDH(CDH(+)) (n=24 for each group). Real-time RT-PCR analysis was performed to investigate pulmonary gene expression levels of LKLF. Differences between the 3 groups at each time point were tested statistically and significance was accepted at p<0.05. Immunohistochemistry was also performed to evaluate LKLF protein expression and distribution.

Results: The relative mRNA expression levels of LKLF on D18 and D21 were significantly decreased (p<0.01) in CDH(−) and CDH(+) groups compared to controls. The gene expression levels of LKLF on D15 did not differ significantly between the nitrofen group and controls. Immunohistochemical study showed strong LKLF immunoreactivity on D18 and D21 in nitrofen-induced hypoplastic lung compared to controls, whereas no difference was seen on D15.

Conclusions: Our results provide evidence for the first time that LKLF is down-regulated in the later stages of lung development in nitrofen-induced hypoplastic lungs. These data suggest that the down-regulation of LKLF during this critical period of lung morphogenesis may impair lung development and maturation, resulting in pulmonary hypoplasia in the nitrofen CDH model.

Key words

lung Kruppel-like factor - nitrofen - hypoplastic lung - congenital diaphragmatic hernia - lung development

References

• 1 Colvin J, Bower C, Dickinson JE. et al . Outcomes of congenital diaphragmatic hernia: a population-based study in Western Australia.  Pediatrics. 2005;  116 356-363
• 2 Stage G, Fenton A, Jaffray B. Nihilism in the 1990s; the true mortality of congenital diaphragmatic hernia.  Pediatrics. 2003;  112 532-535
• 3 Gosche JR, Islam S, Boulanger SC. Congenital diaphragmatic hernia.  Am J Surg. 2005;  190 324-332
• 4 Robinson PD, Fitzgerald DA. Congenital diaphragmatic hernia.  Pediatr Respir Rev. 2007;  8 323-334
• 5 Doi T, Hajduk P, Puri P. Upregulation of Slit-2 and Slit-3 gene expressions in the nitrofen-induced hypoplastic lung.  J Pediatr Surg. 2009;  44 2092-2095
• 6 Doi T, Puri P. Up-regulation of Wnt5a gene expression in the nitrofen induced hypoplastic lung.  J Pediatr Surg. 2009;  44 2302-2306
• 7 Montedonico S, Nakazawa N, Puri P. et al . Congenital diaphragmatic hernia and retinoids: searching for an etiology.  Pediatr Surg Int. 2008;  24 (7) 755-761
• 8 Noble BR, Babiuk RP, Clugston RD. et al . Mechanisms of the congenital diaphragmatic hernia-inducing teratogen nitrofen.  Am J Physiol Lung Cell Mol Physiol. 2007;  293 1079-1087
• 9 Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development.  Am J Physiol Lung Cell Mol Physiol. 2000;  279 1159-1171
• 10 Jay PY, Bielinska M, Erlich JM. et al . Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects.  Dev Biol. 2007;  301 602-614
• 11 Anderson KP, Kern CB, Crable SC. et al . Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Kruppel-like factor: identification of a new multigene family.  Mol Cell Biol. 1995;  15 5957-5965
• 12 Wani MA, Means Jr RT, Lingrel JB. et al . Loss of LKLF function results in embryonic lethality in mice.  Transgenic Research. 1998;  7 229-238
• 13 Wani MA, Wert SE, Lingrel JB. Lung Kruppel-like factor, a zinc finger transcription factor, is essential for normal lung development.  J Biol Chem. 1999;  274 21180-21185
• 14 Kuo CT, Veselits ML, Barton KP. et al . The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis.  Genes Dev. 1997;  11 2996-3006
• 15 Dekker RJ, Boon RA, Rondaij MG. et al . KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium.  Blood. 2006;  107 4354-4363
• 16 Lin Y, Ryan J, Lewis J. et al . TRAF2 exerts its antiapoptotic effect by regulating the expression of Kruppel-like factor LKLF.  Mol Cell Biol. 2003;  23 5849-5856
• 17 Chaudhari BR, Murphy RF, Agrawal DK. Following the TRAIL to apoptosis.  Immunol Res. 2006;  35 249-262
• 18 Sisto M, D’Amore M, Caprio S. et al . Tumor necrosis factor inhibitors block apoptosis of human epithelial cells of the salivary glands.  Ann N Y Acad Sci. 2009;  1171 407-414
• 19 Das H, Kumar A, Lin Z. et al . Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes.  Proc Natl Acad Sci USA. 2006;  103 6653-6658
• 20 Ohshiro K, Miyazaki E, Taira Y. et al . Upregulated tumor necrosis factor-alpha gene expression in the hypoplastic lung in patients with congenital diaphragmatic hernia.  Pediatr Surg Int. 1998;  14 21-24
• 21 Tong QS, Zheng LD, Tang ST. et al . Nitrofen suppresses cell proliferation and promotes mitochondria-mediated apoptosis in type II pneumocytes.  Acta Pharmacol Sin. 2007;  28 672-684

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