Decreased Mitogen Activated Protein Kinase Activities in Congenital Diaphragmatic Hernia–Associated Pulmonary Hypoplasia By David E. Kling, Vinod Narra, Saleem Islam, T. Bernard Kinane, Alessandro Alessandrini, Louis Ercolani, Patricia K. Donahoe, and Jay J. Schnitzer Boston, Massachusetts Background/Purpose: The mechanisms that cause pulmo- nary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). Methods: ERK-1 activities were measured using immune- complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil–treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, acti- vated) forms of the enzymes. Results: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil–treated controls (P  .02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P = .003; dp-ERK-2, P = .04), whereas ERK-1 and ERK-2 protein levels were un- changed. Conclusions: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are re- duced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes. J Pediatr Surg 36:1490-1496. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Congenital diaphragmatic hernia, mitogen activated protein kinase, lung, fetal, nitrofen, extracellular regulated kinase. C ONGENITAL diaphragmatic hernia (CDH) is a lethal birth defect in which the diaphragm fails to form completely in the developing fetus. The frequency of this defect varies from 0.01% to 0.045% of live births, with a 60% mortality rate, caused by inadequate lung function from pulmonary hypoplasia and persistent pul- monary hypertension of the newborn. 1 CDH observed in humans can be modeled in the laboratory by nitrofen administration to pregnant rats and by creation of reti- noic acid-receptor mutants in mice. 2-5 Of these, the rat-nitrofen model has been characterized more exten- sively. 2-4 Administration of nitrofen (2,4-dichlorophe- nyl-p-nitrophenenylether) to pregnant rats at embryonic day 9.5 (E9.5) causes 60% to 90% of the pups to have pulmonary hypoplasia associated with CDH. 2,3 Pulmo- nary immaturity in this model is characterized by re- duced lung weight, lung weight to body weight ratio, disaturated phosphatidylcholine (DSPC) per microgram DNA, and protein per microgram DNA, whereas total DNA and glycogen levels were shown to be higher. 6 In addition, morphometric indices including the total num- ber of air spaces, internal surface area, airspace volume fractions, and duct fractions are lower in CDH compared with controls. 7 Furthermore, in the sheep surgical model of diaphragmatic hernia, similar lung biochemical and morphometric parameters were observed, confirming that the effect produced by nitrofen is characteristic of pulmonary hypoplasia. 8,9 Growth and maturation of the whole lung depend on the ability of each cell to proliferate and differentiate in precise organized patterns in response to extracellular signals. Cell surface–associated molecules including re- ceptor tyrosine kinases (RTKs), G-protein– coupled re- From the Pediatric Surgical Research Laboratories, Pediatric Sur- gical Services, and the Department of Surgery, Laboratory of Devel- opmental Immunology, and Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA. Portions of this work were presented at Experimental Biology ’98, April 18-22, 1998, San Francisco, CA, and it was published in abstract form (Narra V, Islam S, Kinane B, et al: Decreased mitogen-activated protein kinase activity in prenatal rat lungs with congenital diaphrag- matic hernia. FASEB J, 12:A314, 1998 [abstr]) and at Experimental Biology ’99, April 17-21, 1999, Washington, DC, and it was published in abstract form (Kling DE, Schnitzer JJ: Decreased MEK1 enzymatic activity in prenatal rat lungs with congenital diaphragmatic hernia. FASEB J, 13:A354, 1999 [abstr]). Supported by grants from the NIH (1R01HL62615) and the MGH Department of Surgery. Address reprint requests to Jay J. Schnitzer, MD, PhD, Pediatric Surgical Services, Massachusetts General Hospital, 55 Fruit St, WRN 11, Boston, MA 02114-2696. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3610-0004$35.00/0 doi:10.1053/jpsu.2001.27029 1490 Journal of Pediatric Surgery, Vol 36, No 10 (October), 2001: pp 1490-1496