Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis

Abstract

There is growing evidence for the role of CFTR (cystic fibrosis transmembrane conductance regulator) in lung development and differentiation. The mechanism by which the chloride channel could affect lung organogenesis, however, is unknown. In utero CFTR gene transfer in the fetal lungs of mice, rats, and non-human primates was shown previously to alter lung structure and function. A study of the genes altered in the fetal rat lung following CFTR overexpression was initiated in an effort to determine the molecular mechanism for CFTR-dependent differentiation. In utero gene transfer with recombinant adenoviruses carrying either a reporter gene or CFTR resulted in the increased expression of a number of genes upon microarray analysis. The majority of the genes overexpressed in the CFTR-treated lungs were primarily associated with muscle structure and function. Histological and biochemical characterization of these proteins including myosin heavy chain, heat shock protein 27, and isoforms of myosin light chain showed that CFTR overexpression had a profound effect on smooth muscle contraction-related proteins. The CFTR-dependent regulation of smooth muscle contraction related proteins was shown to be related to chloride and extracellular ATP and was dependent upon the PI3 Kinase and Phospholipase C pathways. The changes in smooth muscle proteins were consistent with CFTR-dependent contractions of the embryonic airway smooth muscle. An assay was developed using fluorescent polystyrene beads to show that CFTR did indeed increase amniotic fluid flow into the fetal lung. Increased amniotic fluid pressure was shown previously to be associated with stretch-induced differentiation of the lung. Evaluation of neonatal respiratory function showed that CFTR-dependent muscle contractions and increased amniotic fluid pressure resulted in accelerated maturation of the neonatal rat lung. In addition, these CFTR-dependent changes were shown to be sufficient to reverse the lung phenotype of the CFTR knockout mouse. Mechanical forces influence lung development through pulmonary distension. CFTR overexpression in the fetal lung altered differentiation and development in the lung. These results are consistent with CFTR influencing lung development by regulating the muscle contractions associated with cytoskeletal tension and stretch induced differentiation. Deficiency of CFTR altering lung development would contribute significantly to the Cystic Fibrosis disease phenotype.

Mechanical control of tissue and organ development

“…Fluid shear forces promote kidney development by orchestrating collective epithelial movements to form functional nephrons in zebrafish (Vasilyev et al, 2009). Increased amniotic fluid pressure and cystic fibrosis transmembrane conductive regulator-dependent muscle contractions similarly accelerate lung maturation in rats (Cohen and Larson, 2006), and pressure caused by the inspiration of air with the first breath after birth appears to be crucial for lung maturation, as rat lung epithelium increases surfactant synthesis and secretion when mechanically distorted (Gutierrez et al, 2003).…”

“…Finally, it is important to note that abnormal mechanical environments can interfere with normal organogenesis and produce organ malformations (Bartman et al, 2004;Cohen and Larson, 2006;Hove et al, 2003;Inanlou and Kablar, 2003;Lucitti et al, 2007;Moore et al, 2005;Vasilyev et al, 2009) (Table 1). In addition, certain developmental abnormalities, such as esophageal atresia, a blind-ended pouch of the esophagus, can be repaired clinically solely by restoring normal tension distributions using tensed surgical sutures that mechanically stretch the remaining tissue structures (Foker et al, 1997).…”

“…Mechanical forces are crucial for the formation of blood vessels (Lucitti et al, 2007;Mammoto et al, 2009), lungs (Cohen and Larson, 2006;Gutierrez et al, 2003;Inanlou and Kablar, 2003;Moore et al, 2005), kidneys (Serluca et al, 2002;Vasilyev et al, 2009), muscle (Kahn et al, 2009), mammary glands (Alcaraz et al, 2008), brain (Anava et al, 2009Moore et al, 2009;Wilson et al, 2007), cartilage and bone (Ohashi et al, 2002;Stokes et al, 2002), as well as of the hematopoietic system (Adamo et al, 2009;North et al, 2009) and the heart (Forouhar et al, 2006;Hove et al, 2003;Voronov et al, 2004). For example, in zebrafish embryos, the heart starts pumping blood using a hydroelastic impedance pumping mechanism in which waves of contraction and elastic deformation of the heart chamber, generated by myocytes located near the heart tube entrance, travel forward and reflect back to generate dynamic suction forces that drive blood flow even before valves form (Forouhar et al, 2006).…”

“…Fluid shear forces promote kidney development by orchestrating collective epithelial movements to form functional nephrons in zebrafish (Vasilyev et al, 2009). Increased amniotic fluid pressure and cystic fibrosis transmembrane conductive regulator-dependent muscle contractions similarly accelerate lung maturation in rats (Cohen and Larson, 2006), and pressure caused by the inspiration of air with the first breath after birth appears to be crucial for lung maturation, as rat lung epithelium increases surfactant synthesis and secretion when mechanically distorted (Gutierrez et al, 2003).In the developing mouse heart, nonmuscle myosin heavy chains IIA and IIB are asymmetrically expressed in the embryonic heart tube, and their position appears to be closely correlated with the direction of heart looping regardless of the distribution of the key transcription factor Pitx2 (Linask et al, 2003;Linask et al, 2002;Lu et al, 2008). Moreover, the disruption of myosin-based tension generation with a ROCK inhibitor during the initial stages of heart looping disturbs morphogenesis in this system (Wei et al, 2002); however, tension inhibitors do not interfere with heart looping in chicks, so the conservation of this mechanism remains uncertain (Remond et al, 2006).…”

“…The abnormal Rho-mediated sensing of mechanical cues in the tumor microenvironment also appears to contribute to the aberrant behavior of tumor capillary endothelial cells, resulting in the development of characteristic structural abnormalities in the cancer microvasculature that interfere with drug delivery and radiation therapy (Ghosh et al, 2008). Taken together, these findings suggest that cancer could be seen as a disease of tissue development that results from the deregulation of both chemical and mechanical cues in the local tissue microenvironment.Finally, it is important to note that abnormal mechanical environments can interfere with normal organogenesis and produce organ malformations (Bartman et al, 2004;Cohen and Larson, 2006;Hove et al, 2003;Inanlou and Kablar, 2003;Lucitti et al, 2007;Moore et al, 2005;Vasilyev et al, 2009) (Table 1). In addition, certain developmental abnormalities, such as esophageal atresia, a blind-ended pouch of the esophagus, can be repaired clinically solely by restoring normal tension distributions using tensed surgical sutures that mechanically stretch the remaining tissue structures (Foker et al, 1997).…”