Page 1 of 32 in PresS. Am J Physiol Regul Integr Comp Physiol (March 30, 2006). doi:10.1152/ajpregu.00880.2005 Articles R-00880-2005.R1
Uteroplacental insufficiency increases p53 phosphorylation without triggering the p53-MDM2 functional circuit response in the IUGR rat kidney. Mariana Baserga, Merica A. Hale, Xingrao Ke, Zeng Ming Wang, Xing Yu, Christopher W. Callaway, Robert A. McKnight, Robert H. Lane
University of Utah School of Medicine, Department of Pediatrics, Division of Neonatology, Salt Lake City, UT, 84158.
Running Head: Kidney p53-MDM2 Functional Circuit in Newborn IUGR rats.
Contact Information: Mariana Baserga, MD University of Utah School of Medicine Department of Pediatrics Division of Neonatology PO Box 581289 Salt Lake City, UT 84158 P: 801-587-3005 F: 801-585-7395 E-mail:
[email protected]
1 Copyright © 2006 by the American Physiological Society.
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ABSTRACT Uteroplacental insufficiency (UPI) leads to intrauterine growth restriction (IGUR) which predisposes infants towards renal insufficiency early in life and increases the risk of kidney related adult morbidities such as hypertension. This compromised in utero environment has been demonstrated to impair nephrogenesis, as evidenced by a reduced nephron endowment in humans and in rats rendered IUGR by UPI. Concordantly, we have observed that IUGR rats have increased kidney p53 protein levels associated with increased apoptosis. Several factors can regulate p53 gene expression and activity, including posttranslational modifications and protein-protein interactions in the cell. Among these, 2 important mechanisms are: 1) phosphorylation of the amino terminal serine 15 [phospho-p53 (Ser15)] which increases p53 stability and apoptotic activity, and 2) the murine double minute (MDM2) functional circuit which functions to limit further p53-induced apoptosis by promoting proteosomal degradation of p53. We hypothesize that UPI induces an increase in phospho-p53 (Ser15) in association with an absent MDM2 response, predisposing the kidney to increased apoptosis. To test our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. UPI significantly increased phospho-p53 (Ser15) as well as ATM/ATR and DNA-PK kinase levels, which induce phosphorylation of p53. In contrast, UPI induced no increase in kidney MDM2 mRNA and protein levels in IUGR pups. We conclude that among multiple mechanisms that affect nephrogenesis, UPI induces an increase in p53 phosphorylation without a corresponding increase in MDM2 expression and speculate that this response may contribute to the increased apoptosis previously described in the IUGR kidney.
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Key words: ATM/ATR, DNA-PK, nephrogenesis, apoptosis.
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INTRODUCTION Intrauterine growth restriction (IUGR) caused by uteroplacental insufficiency (UPI) is a morbidity associated with several common complications of pregnancy such as preeclampsia and maternal diabetes (57). Epidemiological studies have demonstrated that IUGR neonates experience increased morbidity and mortality rates, including impaired renal function, and are at increased risk to develop adult morbidities, such as hypertension (12, 27, 51, 53). In chronic fetal hypoxia, such as seen in UPI, peripheral blood flow and blood flow to the kidneys is reduced to maintain brain, heart and adrenal perfusion (39). This is known as the brain-sparing effect and leads to a condition termed “asymmetrical growth restriction”. Interestingly, both human and animal studies have shown that IUGR results in smaller kidneys with decreased nephron number (3, 8, 49, 51). UPI affects multiple components of the fetal milieu, so a single mechanism responsible for abnormal kidney development is unlikely. For instance, nephrogenesis involves rapid remodeling of structures which requires massive apoptosis, making this is an important and active mechanism of normal human and rodent fetal kidney development (24). Our laboratory has previously demonstrated that rats rendered IUGR by bilateral uterine artery ligation also suffer reduced nephron number, in association with increased apoptosis (41). A key regulator of apoptosis is p53, which acts as both an active component of the apoptosis cascade and a transcription factor (19, 46). In response to stress, hypoxia, and damaged DNA, p53 accumulates in the cell nucleus and is activated as a transcription factor (4). Activation of p53 initiates or inhibits the expression of numerous genes that mediate cell cycle arrest or induce apoptosis (5). Many of the initial insults that characterize our
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model of UPI and IUGR, including moderate hypoxia, acidosis, hypoglycemia, and decreased levels of key growth factors such as IGF-I, can potentially increase p53 expression in different organs, including the kidney (9, 18, 44, 52). Indeed, in our IUGR rat animal model, kidney p53 protein levels are increased at birth. Furthermore, the expression of apoptosis-related molecules, Bcl-2 and Bax, are also affected in the kidney of these IUGR pups. Bcl-2 and Bax contribute to the signaling pathway that activate caspase-3, which is necessary for the chromatin condensation and DNA cleavage that characterize apoptosis. Through its function as a transcription factor, p53 negatively regulates Bcl-2 expression (antiapoptotic protein) and positively regulates Bax (proapoptotic protein) expression (48, 59). However, the mechanisms underlying the change in p53 protein levels in the IUGR kidney are not fully understood. In the present study we focus on the signaling pathways and molecular mechanisms underlying the increase in p53 levels and apoptosis observed in the IUGR rat kidney. Several factors can regulate p53 gene expression and activity, including posttranslational modifications and protein-protein interactions in the cell. Among these factors, two important mechanisms regulate p53 protein levels and activity. The first one includes posttranslational modification such as phosphorylation of the amino terminal serine 15 [phospho-p53 (Ser15)] which increases p53 stability and apoptotic activity (4). Several kinases have been identified that detect cellular stress and initiate signaling pathways through phosphorylation of p53 at Ser 15. These include the ataxia teleangiectasia mutated (ATM) kinase, A-T related (ATR) kinase, and dsDNAactivated protein kinase (DNA-PK) kinase (4, 40).
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The second mechanism that controls p53 levels and activity involves murine double minute (MDM2), which functions to limit further p53-induced apoptosis (47). MDM2, a p53 specific E3 ubiquitin ligase, is the principal cellular antagonist of p53, blocking the interaction of p53 with transcriptional co-activators and promoting p53 proteosomal degradation (34, 54). p53 and MDM2 form one feedback loop termed “the p53 functional circuit”, in which p53 positively regulates MDM2 by activating the gene mdm2 transcription, and MDM2 negatively regulates p53 by promoting p53 ubiquitination and degradation (21). Posttranslational modification of the MDM2 protein by phosphorylation at serine 166 [phospho-MDM2 (Ser166)] increases MDM2 function by increasing nuclear entry (60). MDM2 enters the nucleus to form the “p53-MDM2 complex” that then shuttles to the cytoplasm where p53 degradation takes place. The goal of this autoregulatory negative feedback loop is to maintain low levels of p53 in the absence of stress, and to limit the severity of the p53 response to cellular stress. Based on the fact that UPI predisposes to increased kidney apoptosis both in humans and animals, we hypothesized that IUGR induces an increase in p53 phosphorylation at Ser15 through elevated ATM, ATR and DNA-PK kinases. Furthermore, we hypothesized that the p53-MDM2 functional circuit fails to respond to the UPI insult in the immature kidney, leading to an absent MDM2 response, and therefore, increased apoptosis. To prove our hypothesis, bilateral uterine artery ligation (IUGR) and sham surgery (SHAM) were performed on day 19 of gestation in Sprague-Dawley rats (term, 21.5 days). Similar to the human, UPI results in offspring with low birth weight and asymmetrical IUGR (50). In this well-established model of asymmetrical growth
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restriction, IUGR pups are 20-25% lighter than the sham-operated control animals at birth (IUGR: 4.00 ± .25 vs. sham: 5.25 ± .22, p
