Differential expression of cyclic nucleotide phosphodiesterases 4 in developing rat lung

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During the perinatal period, lungs undergo changes to adapt to air breathing. The genes involved in these changes are developmentally regulated by various signaling pathways, including the cyclic nucleotide cAMP. As PDE4s are critical enzymes for
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  1 Differential Expression of Cyclic Nucleotide Phosphodiesterases 4 in Developing Rat Lung Running Title: PDE4 in developing rat lung Emmanuel Lopez 1,2,3,4 , Pierre-Henri Jarreau 1,2,3,4 , Elodie Zana 1,3 , Marie-Laure Franco-Montoya 3,5 , Thomas Schmitz 1,2,3,6 , Danièle Evain-Brion 1,3 , Jacques Bourbon 3,5 , Christophe Delacourt 3,5 , Céline Méhats 1,3,*   1 UMR 767 Inserm-Paris Descartes, Paris, France;  2 Université Paris Descartes, Faculté de Médecine, Paris, France; 3 PremUP, Paris, France; 4 AP-HP, Groupe hospitalier Cochin-Saint Vincent de Paul, Service de Médecine Néonatale de Port-Royal, Paris, France; 5 Inserm U955, Créteil, France; 6 AP-HP, Groupe hospitalier Cochin-Saint Vincent de Paul, Service d’Obstétrique, Maternité de Port -Royal, Paris, France. * Corresponding author: Céline Méhats, UMR 767 Inserm-Paris Descartes, Hôpital Saint Vincent de Paul, 82 Avenue Denfert-Rochereau, 75014 Paris, France. E-mail address: celine.mehats@inserm.fr Phone: 33 1 40 48 82 38, Fax: 33 1 40 48 83 94 Statement of financial support: This work was supported by grants from la Chancellerie des Universités de Paris (Legs Poix 2006) and from Air Liquide Foundation.      h  a   l  -   0   0   5   2   6   9   0   1 ,  v  e  r  s   i  o  n   1  -   1   6   O  c   t   2   0   1   0 Author manuscript, published in "Developmental Dynamics 239, 9 (2010) 2470-8" DOI : 10.1002/dvdy.22374  2 Abstract  During the perinatal period, lungs undergo changes to adapt to air breathing. The genes involved in these changes are developmentally regulated by various signaling pathways, including the cyclic nucleotide cAMP. As PDE4s are critical enzymes for regulation of cAMP levels, the objective of this study was to investigate PDE4s ontogeny in developing rat lung during the perinatal period. Pulmonary PDE4 activity, PDE4A-D and PDE4B and PDE4D variants expression levels, PDE4B and PDE4D protein levels, and PDE4Ds localization in distal lung were determined. PDE4 activity increased towards term, dropped at birth, and increased thereafter to reach a plateau at the end of the second week of life. PDE4B2 and PDE4D long forms demonstrated a pattern of expression that increased markedly at birth. After birth, PDE4D were expressed in alveolar epithelial and mesenchymal cells. The study therefore evidenced striking variations in expression patterns among the PDE4 family that differed from changes in global PDE4 activity. Key words:  lung development, phosphodiesterases 4, cyclic AMP    h  a   l  -   0   0   5   2   6   9   0   1 ,  v  e  r  s   i  o  n   1  -   1   6   O  c   t   2   0   1   0  3 Introduction  Successful transition to air breathing at birth involves dramatic changes in lung  physiology, including differentiation of alveolar type II cells, clearance of pulmonary fluid, decrease of pulmonary vascular resistance, and surfactant release (Cardoso, 2001). Defects in these physiological changes may affect neonates and can be responsible for neonatal respiratory disorders, which represent the leading cause of hospitalization in neonatal intensive care units. Preterm infants may develop bronchopulmonary dysplasia (BPD), a disease mainly related to impairment of alveolarization and microvascular development. Identification of the molecular and physiological processes controlling perinatal lung development and alveolarization would be promising to explore and develop new therapeutic approaches to cure or prevent respiratory disorders. Lung adaptation to air breathing and lung development are controlled by a large number of factors. Many of these factors involve signaling pathways involving cAMP (Mendelson, 2000). Earlier studies have focused on developmental changes in cAMP/PKA pathway as key controllers of the actions of catecholamines or other hormones in perinatal lungs. Manipulation of the cAMP pathway facilitated surfactant release and reduced surface tension in experimental models (Aeberhard et al., 1984; Odom et al., 1987; Fisher et al., 1991). Ontogenic variations in PKA and some of its protein substrates have been described: the  phosphorylation pattern and the expression of specific phosphatase or adenylate cyclase activities changed during lung development (Whitsett et al., 1982a; Whitsett et al., 1982b; Whitsett et al., 1983; Whitsett et al., 1985; Acarregui et al., 1998). Cyclic nucleotide phosphodiesterases (PDE) represent a superfamily of enzymes that hydrolyze cAMP. Eleven families of PDE have been described in mammals, and selective inhibitors of specific families are now used or proposed to treat various disorders including cardiovascular disease, erectile dysfunction, pulmonary arterial hypertension, and chronic    h  a   l  -   0   0   5   2   6   9   0   1 ,  v  e  r  s   i  o  n   1  -   1   6   O  c   t   2   0   1   0  4  pulmonary disorders (Bender and Beavo, 2006). Among these families, the PDE4 family has  been extensively investigated over recent decades for its potential as a new pharmacological target in asthma and chronic obstructive pulmonary disease in adults (Boswell-Smith and Spina, 2007; Spina, 2008). PDE4s are encoded by four genes designated A through D, and encompass more than 20 different proteins produced through differential promoter activation (Conti et al., 2003). Because of this diversity, each PDE4 protein is now thought to have a specific, non-redundant function in controlling cAMP metabolism within physiological processes (Conti et al., 2003). The ontogeny and precise role of PDE4s in the perinatal developing lung and alveolarization have not yet been investigated. Rat pups are a common experimental model to study normal or pathological distal lung development. They are born at the saccular phase of lung development and alveolarization is an entirely postnatal event, starting on the fourth postnatal day and ending at day 21. The potential of PDE4 inhibition was evaluated in an experimental model of BPD in rat pups and demonstrated that PDE4 inhibition may impair alveolarization (Mehats et al., 2008). Although a global impact via a decrease of somatic growth is certainly involved in this deleterious effect, the possibility of a specific involvement of PDE4 proteins in lung development must also be considered. The present study investigated global PDE4 activity and specific PDE4 protein expression patterns in fetal and newborn rats. Specific isoforms of PDE4B and PDE4D varied  profoundly during distal lung development, suggesting a role for these proteins in perinatal lung physiology.    h  a   l  -   0   0   5   2   6   9   0   1 ,  v  e  r  s   i  o  n   1  -   1   6   O  c   t   2   0   1   0  5 Results PDE4 activity in lung during fetal and early postnatal life At fetal day 17 (F17, term F22), PDE4 activity was 14±2 pmol/min/mg and increased significantly towards term, doubling at F21 (28±3 pmol/min/mg) (Figure 1). After birth, PDE4 activity decreased transiently (16±2 pmol/min/mg) before increasing sharply from P7 (44±7 pmol/min/mg), reaching a plateau at P14 (60±9 pmol/min/mg). No gender effect was observed (at P1 males: 18±1 pmol/min/mg, n=5; females: 17±4 pmol/min/mg, n=6; other data not shown). Pulmonary PDE4 mRNA levels during fetal and early postnatal life Developmental changes in mRNA levels of the four PDE4 genes were investigated using generic primers covering all known variants derived from each gene. Data are expressed as  Ct, which means that a variation of 1 cycle represents a 2-fold increase or decrease and that a low  Ct indicates high expression. At F17, PDE4A and PDE4B mRNAs were the most abundant forms, with 8-fold lower and 1,000-fold lower expression of PDE4D and PDE4C, respectively (Table 2). PDE4C remained the PDE4 with the lowest expression at all stages examined, although PDE4C mRNA increased significantly after F21 (Figure 2). PDE4A mRNA expression levels did not change significantly either during saccular and alveolar stages, or at birth. PDE4B and PDE4D mRNAs rose at F21, peaked on P1 to reach 25- and 60-fold their F17 levels, respectively, then decreased 2- to 3-fold at the beginning of alveolarization and decreased further at P21 (Figure 2). PDE4B and PDE4D protein levels in developing rat lung Subsequent studies focused on PDE4B and PDE4D variant protein expression. Using PDE4B-specific antibodies that recognized the common C-terminus of all PDE4B isoforms, a    h  a   l  -   0   0   5   2   6   9   0   1 ,  v  e  r  s   i  o  n   1  -   1   6   O  c   t   2   0   1   0
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