View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Genomics 88 (2006) 173–184 www.elsevier.com/locate/ygeno Correlation of serpin–protease expression by comparative analysis of real-time PCR profiling data Sunita Badola a, Heidi Spurling a, Keith Robison a, Eric R. Fedyk a, Gary A. Silverman b, ⁎ Jochen Strayle c, Rosana Kapeller a,1, Christopher A. Tsu a, a Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, USA b Department of Pediatrics, University of Pittsburgh School of Medicine, Magee-Women’s Hospital, 300 Halket Street, Pittsburgh, PA 15213, USA c Bayer HealthCare AG, 42096 Wuppertal, Germany Received 2 December 2005; accepted 27 March 2006 Available online 18 May 2006 Abstract Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin–protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin–protease matched expression may underlie the imbalanced protease activity observed in pathological states. © 2006 Elsevier Inc. All rights reserved. Keywords: Protease; Serpin; Cancer; Imbalance; Expression profiling; Gene correlation; PCR Proteases play critical roles in many normal processes such We have chosen to study the largest family of endogenous as development, coagulation, and immunity. Therefore, protease inhibitors, the serpins, which target a wide variety of regulatory mechanisms must exist to ensure that homeostasis serine and cysteine proteases [2,3]. Over 1000 serpins have is maintained. In this regard, there are endogenous inhibitors been identified across all species, including 37 human forms for most of the known protease families [1]. A protease and [4]. Their function is mediated by a unique suicide substrate its cognate inhibitor may normally be closely matched in site mechanism, in which cleavage of an exposed substrate-like and amount of expression. An imbalance in expression sequence (reactive site loop, RSL) causes a dramatic confor- patterns may arise as either a causative or responsive element mational change in the serpin, thereby trapping the protease in a in disease. covalently bound, inactive state [5]. The first known serpin, α1- antitrypsin (SERPINA1), is a secreted glycoprotein that is found in high levels in the serum [6]. It is produced primarily in the Abbreviations: serpin, serine protease inhibitor; RSL, reactive site loop; UUI, uterine urinary incontinence; BM-MNC, bone marrow mononuclear cells; liver, but also by a few inflammatory and epithelial cell types IBD, inflammatory bowel disease; DRG, dorsal root ganglion; CHF, chronic [7]. A primary site of action is the lung, where SERPINA1 heart failure; COPD, chronic obstructive pulmonary disease; PBMC, peripheral inhibits human neutrophil elastase, preventing tissue damage. blood mononuclear cells; BPH, benign prostate hyperplasia; SMC, smooth Hereditary loss of SERPINA1 has been implicated in chronic β muscle cell; B2M, 2-microglobulin; siRNA, small interfering RNA. liver disease and emphysema, as well as an increased risk of ⁎ Corresponding author. Fax: +1 617 551 8906. E-mail address: [email protected] (C.A. Tsu). liver and other cancers [6,8]. Conversely, elevated plasma levels 1 Current address: Renegade Therapeutics, One Broadway, 14th floor, of the serpin squamous cell carcinoma antigens SCCA1 and 2 Cambridge, MA 02142, USA. (SERPINB3 and SERPINB4) are an established marker for this 0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.03.017 174 S. Badola et al. / Genomics 88 (2006) 173–184 disease [9,10]. Although they interact in vitro with cathepsins S, Particular attention was paid to disease relevance and site L, and K, or cathepsin G and chymase, respectively, the of expression in the selection of serpins. This resulted in the biological roles of SERPINB3 and SERPINB4 remain un- selection of most of the intracellular ov-serpins (clade B) [2]. known [11,12]. Here we suggest that expression profiling Although an effort was made to avoid secreted serpins, as site correlation may aid in the identification of novel in vivo of action may not correlate well with site of expression, we functions of serpins and potential target proteases. did select a small number of secreted serpins (clades A, E, We utilized real-time PCR (TaqMan PCR) to determine the and I), based primarily on biochemical characterization and expression profiles of 20 serpins and 105 serine and cysteine disease biology. Several novel, previously uncharacterized proteases across a panel of 58 samples from a collection of 13 serpins were also selected. In total, 20 serpins (Table 1) were cell lines and 31 normal and 14 diseased human tissues. In chosen such that the method could be validated with known general, highly restricted expression of serpins was observed interactions and that new serpin–protease pairs might be in normal and diseased tissues, suggesting defined physio- identified de novo. logical roles for these protease inhibitors. Furthermore, in specific cases, the expression correlations observed in lung, Serpin expression profile ovarian, breast, and prostate cancer tissues were altered compared to normal tissues. The expression profile of 20 serpins across 58 samples from a collection of 13 cells and 31 normal and 14 diseased human tissues Results is depicted in Fig. 1A. The normalized relative expression is represented by a light to dark color grid, which shows low to high Protease and serpin selection criteria levels of mRNA transcripts. For example, SERPINB1 and SERPINB8 are ubiquitously expressed, whereas SERPINI2 is The entire human genomic complement of proteases (greater expressed only in pancreas. In general, good agreement was found than 500) and serpins (37) is relatively large [1,13]. Criteria between the serpin mRNA expression patterns in this work and were therefore used to select a subset for transcription profiling. previously reported results (mRNA and/or protein). This would Factors included known disease link, known target or substrate, include serpins A1, A3, and A4 (liver, pancreas, and kidney) site of expression (tissue, intracellular or secreted), protein [7,14,15]; A5 (pancreas, kidney, liver, and ovary) [16];B8 structure (fold, essential catalytic or inhibitory residues), and (digestive tract, lung, liver, pituitary, skin, and tonsil) [17];I1 homology to proteins of known activity or disease relevance. (brain cortex, hypothalamus, pituitary, and spinal cord) [18];andI2 Aspartyl and metalloproteases were removed from consider- (pancreas) [19]. The most striking and unexpected finding is the ation, as serine (trypsin, subtilase) and cysteine proteases restricted expression of most serpins. The majority of the serpins are account for the majority of proteases known to interact with expressed at “high” levels in four or fewer, often related, tissues in serpins. In this way, a total of 81 trypsin, 10 subtilase, and 14 our panel (Fig. 1A). This supports the hypothesis that serpin cysteine proteases were selected for profiling (Supplemental expression is highly regulated and that imbalanced expression may Table 1). lead to pathological states. Table 1 Serpin sequences Nomenclature GenBank Common name RSL (P1/P1′) Putative targets SERPINA1 X01683 α1-Antitrypsin GTEAAGAMFLEAIPM/SIPPE Trypsin, neutrophil elastase SERPINA3 X68733 α1-Antichymotrypsin GTEASAATAVKITLL/SALVE Cathepsin G, chymase, chymotrypsin SERPINA4 L19684 Kallistatin GTEAAAATSFAIKFF/SAQTN Kallikrein SERPINA5 M68516 Protein C inhibitor GTRAAAATGTIFTFR/SARLN Thrombin, acrosin SERPINA9 AX392967 Sequence 9 from Patent WO0214358 GTEATAATTTKFIVR/SKDGP Unknown SERPINA11 BD130048 Human serine protease and serpin polypeptide GTEAGAASGLL/SQPPSLNTM Unknown SERPINA12 AX192197 Sequence 13 from Patent WO0149729 GTEGAAGTGAQTLPM/ETPLV Unknown SERPINA13 AX163789 Sequence 1 from Patent WO0138534 GSEAAAATSIQL/TPGPRPDL Unknown SERPINB1 M93056 Leukocyte elastase inhibitor GTEAAAATAGIATFC/MLMPE Neutrophil elastase, cathepsin G SERPINB2 J03603 Plasminogen activator inhibitor-2 GTEAAAGTGGVMTGR/TGHGG uPA SERPINB4 U19557 Squamous cell carcinoma antigen 2 GVEAAAATAVVVVEL/SSPST Cathepsin G, chymase SERPINB7 AF027866 Megsin GTEATAATGSNIVEK/QLPQS Plasmin SERPINB8 L40377 Cytoplasmic antiproteinase 2 GTEAAAATAVVRNSR/CSRME Trypsin, furin SERPINB10 U35459 Bomapin GTEAAAGSGSEIDIR/IRVPS Trypsin, thrombin SERPINB11 AF419954 Epipin GTEAAAATGDSIAVK/SLPMR Unknown SERPINB12 AF411191 Yukopin GTQAAAATGAVVSER/SLRSW Trypsin SERPINB13 AF169949 Hurpin, headpin GTEAAAATGIGFTVT/SAPGH Cathepsins L, K SERPINE3 AX407123 Sequence 5 from Patent WO0226981 GTKASGATALLLLKRSR/IPI Unknown SERPINI1 Z81326 Neuroserpin GSEAAAVSGMIAISR/MAVLY Trypsin, tPA SERPINI2 BC027859 Pancpin GSEAATSTGIHIPVIM/SLAQ