Activating Transcription Factor 3 Is Up-Regulated in Patients with Hypospadias

0031-3998/05/5806-1280 PEDIATRIC RESEARCH Copyright © 2005 International Pediatric Research Foundation, Inc. Vol. 58, No. 6, 2005 Printed in U.S.A. Activating Transcription Factor 3 Is Up-Regulated in Patients with Hypospadias BENCHUN LIU, ZHONG WANG, GUITING LIN, KORAY AGRAS, MICHELE EBBERS, EMILY WILLINGHAM, AND LAURENCE S. BASKIN Center for the Study and Treatment of Hypospadias, Department of Urology [B.L., G.L., K.A., M.E., E.W., L.S.B.], University of California, San Francisco, San Francisco, CA, 94143; Department of Urology, Ninth People’s Hospital [Z.W.], Medical College of Shanghai Jiaotong University, China 200011 ABSTRACT Hypospadias is a congenital anomaly of the genitalia characterized by abnormalities of the urethra and foreskin, with the urethral meatus located in an abnormal position anywhere from the distal ventral penile shaft to the perineum. Because the incidence of hypospadias is approximately 1/200 –1/300 live male births, it is one of the most common congenital malformations, but its etiology is largely uncharacterized. Genomic analysis of hypospadic tissue indicated a potential role for activating transcription factor 3 (ATF3) in the development of this anomaly. ATF3 may be involved in homeostasis, wound healing, cell adhesion, or apoptosis, and normally it is expressed at a steadystate in quiescent cells. Additionally, it has been shown to be an estrogen-responsive gene, and the etiology of hypospadias may be related to in utero exposure to estrogenic or anti-androgenic compounds. We examined the expression of ATF3 in tissues from 28 children with hypospadias compared with 20 normal penile skin tissue samples from elective circumcision. Eighty-six Hypospadias is a congenital anomaly of the genitalia characterized by abnormalities of the urethra and foreskin (1). In mild hypospadias the urethral meatus is located in an abnormal position along the distal ventral penile shaft, coronal margin, or proximal glans. In moderate hypospadias the abnormal urethral opening exists on the middle of ventral aspect of the penis. Severe hypospadias is characterized by a proximal urethral opening that can occur at the penile scrotal junction or within the scrotal folds or perineum, and the more severe forms of hypospadias are associated with penile curvature. The incidence of hypospadias is approximately 1/200⬃1/300 live male births, making hypospadias one of the most common congenital malformations in children. Some research has documented that the incidence of this anomaly has been increasing in the Received November 23, 2004; accepted May 2, 2005. Correspondence: Laurence S. Baskin, Ph.D., Department of Urology, University of California San Francisco, San Francisco, CA; email: This study was supported by NIH grant DK 058105-01 and the American Foundation of Urologic Disease. DOI: 10.1203/01.pdr.0000187796.28007.2d percent of the hypospadias samples were immunohistochemically positive, compared with 13% of normal tissue samples. Seventy-five percent of hypospadias samples were positive from in situ hybridization, compared with 1% of circumcision samples. Our results indicate that ATF3 is up-regulated in the penile skin tissues of boys with hypospadias, suggesting a role for this transcription factor in the development of this abnormality. Because the etiology of hypospadias may include exposure to estrogenic compounds, the responsiveness of ATF3 to estrogen is also discussed. (Pediatr Res 58: 1280–1283, 2005) Abbreviations ATF3, activating transcription factor 3 bZIP, basic region and leucine zipper CREB, cAMP responsive element binding IHC, immunohistochemistry ISH, in situ hybridization United States during the last three decades (2). The molecular events required in the genitourinary tract for normal development of the male external genitalia are just beginning to be elucidated (3-6), and the etiology of hypospadias remains unknown, although endocrine disruptors have been proposed as a possible explanation for the increasing incidence that has been reported in industrialized countries (4 –7). Many studies also suggest a genetic component in the transmission of this birth defect and it seems to be multifactorial (8,9). Activating transcription factor 3 (ATF3) is a member of the ATF/CREB (cAMP responsive element binding) family of transcription factors (10). ATF3 contains the basic region and leucine zipper (bZIP) motif characteristic of the bZIP superfamily of transcription factors, which includes members of the CCAAT/enhancer-binding protein family, the Jun/Fos family and ATF/CREB family (10 –12). Expression of ATF3 protein is normally at a steady state in quiescent cells, but can be rapidly induced to a high level in response to multiple extracellular signals, including growth factors, cytokines, and genotoxic stress agents (10,13–17). 1280 1281 ATF3 UPREGULATED IN HYPOSPADIAS ATF3 may be involved in homeostasis, wound healing, cell adhesion, cancer cell invasion, apoptosis and signaling pathways (10,11,18 –20). Overexpression of ATF3 protein suppresses cell growth and slows the transition of cells from G1 to S phase. This evidence suggests that the ATF3 protein may play a negative role in cell cycle progression (20 –23). Microarray results from our lab (Z. Wang et al., manuscript in preparation) indicate that ATF3 is one of the genes that is up-regulated in hypospadic tissue. Exploration of the literature revealed a potential response of ATF3 to estrogen (9,24), and estrogenic or anti-androgenic activity is implicated in the development of hypospadias (4,25). To investigate further the role of ATF3 in this anomaly, we compared the gene’s mRNA/ protein expression in normal versus hypospadic human tissue using molecular localization techniques. MATERIALS AND METHODS Young males with hypospadias scheduled to undergo surgical repair were prospectively entered into this study from January, 2004 to June, 2004. We received written, informed consent from parents for these procedures, which were approved by our institutional Committee on Human Research. Patients with an undescended testis, intersex condition, or known endocrine abnormalities were excluded from the study. The position of the urethral meatus (Table 1), associated anomalies, and family history of hypospadias were assessed by history and physical exam. No patients received preoperative testosterone treatment. Excess prepucial tissue was obtained at the time of elective surgery for hypospadias; no periurethral tissue was taken. Controls consisted of patients undergoing elective circumcision, and controls and hypospadias samples were strictly age-matched. Tissue specimens were prepared for paraffin-embedded or frozen sectioning. Antibodies and immunohistochemical techniques. Skin specimens were evaluated for expression of ATF3 protein by immunohistochemical staining using the standard protocol accompanying the ABC kit (Vector Laboratories, Inc., CA), with overnight primary-antibody incubat (...truncated)