Central Role for Endothelial Human Deneddylase-1/SENP8 in Fine-Tuning the Vascular Inflammatory Response This information is current as Stefan F. Ehrentraut, Douglas J. Kominsky, Louise E. of September 30, 2021. Glover, Eric L. Campbell, Caleb J. Kelly, Brittelle E. Bowers, Amanda J. Bayless and Sean P. Colgan J Immunol 2013; 190:392-400; Prepublished online 3 December 2012; doi: 10.4049/jimmunol.1202041 Downloaded from http://www.jimmunol.org/content/190/1/392 Supplementary http://www.jimmunol.org/content/suppl/2012/12/03/jimmunol.120204 Material 1.DC1 http://www.jimmunol.org/ References This article cites 53 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/190/1/392.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 30, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Central Role for Endothelial Human Deneddylase-1/SENP8 in Fine-Tuning the Vascular Inflammatory Response Stefan F. Ehrentraut, Douglas J. Kominsky, Louise E. Glover, Eric L. Campbell, Caleb J. Kelly, Brittelle E. Bowers, Amanda J. Bayless, and Sean P. Colgan A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective ther- apies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabi- lization of NF-kB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-a by assessing Cul neddylation status, NF-kB and HIF-1a stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-kB, stabilization of HIF-1a, and increased NF-kB/HIF-a promoter activity in response to LPS. HMECs Downloaded from lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-kB or HIF-1a. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-kB responses, induced HIF-1a promoter activity, and reduced secretion of TNF-a–elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation. The Journal of Immunology, http://www.jimmunol.org/ 2013, 190: 392–400. epsis remains one of the leading causes of death in de- lized, translocates to the nucleus, and enhances transcription of a veloped countries (1, 2). The underlying mechanisms of number of anti-inflammatory genes (13). S sepsis severity are incompletely understood, and therefore The ubiquitination of IkB kinase and the a subunit of HIF-1 treatment options beyond those available today are needed. One of and HIF-2 is mediated through Cullin (Cul) proteins, which, to be the hallmarks of sepsis is loss of endothelial function and subse- active, require conjugation to the small ubiquitin-like modifier quent activation of the immune system (3, 4). The NF-kB pathway neural precursor cell–expressed, developmentally downregulated has been well established as one of the key pathways for in- (Nedd)8 (14). The conjugation of Nedd8 to its target proteins is by guest on September 30, 2021 tracellular proinflammatory signaling (5–7). Furthermore, there a multistep process. Initially, the Nedd8 precursor is cleaved at is evidence that NF-kB can influence hypoxia-inducible factor a carboxyl-terminal glycine residue and conjugated to the UBA3- (HIF), an oxygen homeostasis transcription factor, levels and vice APPB1 E1 ligase, also referred to as Nedd8-activating enzyme versa (8, 9). NF-kB is regulated through IkB kinases and its ac- (15–17). The mature Nedd8 moiety is transferred to its E2 ligase tivation relies on IkB kinase degradation through the 26S pro- ubiquitin–conjugating enzyme 12 (18) and then linked to its E3 teasome (10). HIF is also tightly regulated in a posttranslational ligase, a Cul protein, thereby activating it (19, 20). Nedd8 con- fashion. Under normoxic conditions, HIF is hydroxylated by jugation to Cul-1 is necessary to process the NF-kB p50 subunit prolyl hydroxylases, followed by ubiquitination and ultimately precursor p105 through the Skp-Cul-1-FboxbTrCP (SCFbTrCP) degradation by the proteasome (11). Hypoxia is often detectable (21). Based on their findings, Amir et al. (21) concluded that ned- at sites of inflammation (12). Under such conditions, HIF is stabi- dylation of Cul proteins is essential for induced processing of p105 and NF-kB activity. Apart from NF-kB, Cul neddylation also impacts the transcription factor HIF. The a subunit of HIF, under Mucosal Inflammation Program, Department of Medicine and Immunology, Univer- normoxic conditions, is hydroxylated through prolyl hydroxylases sity of Colorado School of Medicine, Aurora, CO 80045 (22). This hydroxylation facilitates recognition and binding of Received for publication July 24, 2012. Accepted for publication October 29, 2012. HIF-a by the von Hippel–Lindau protein (pVHL), which is part This work was supported by Deutsche Forschungsgemeinschaft Grant EH 371/1-1, National Institutes of Health Grants DK50189, HL60569, and DK095491, and by the of an E3/ligase complex containing Cul-2 (23), thereby medi- Crohn’s and Colitis Foundation of America. ating the proteasomal degradation of hydroxylated HIF (11). In S.F.E. and S.P.C. designed research; S.F.E., D.J.K., L.E.G., E.L.C., C.J.K., B.E.B., its nonhydroxylated form, HIF-a translocates to the nucleus, binds and A.J.B. performed research; and S.F.E., and S.P.C. analyzed data and wrote the to the constitutively expressed HIF-1b subunit, and transcriptionally manuscript. regulates a wide variety of genes important for the inflammatory Address correspondence and reprint requests to Dr. Sean P. Colgan, University of response (24, 25). Colorado School of Medicine, 12700 East 19th Avenue, MS B-146, Aurora, CO 80045. E-mail address: [email protected] Lately some interest has been generated in understanding the The online version of this article contains supplemental material. mechanisms that regulate Cul neddylation, more specifically the Abbreviations used in this article: Cul, Cullin; HIF, hypoxia-inducible factor; HMEC, reverse process, deneddylation. It has been shown that adeno- human microvascular endothelial cell; HRE, hypoxia response element; Nedd, neural sine generated by hypoxic preconditioning mediates deneddylation precursor cell–expressed, developmentally downregulated; pVHL, von Hippel– of Cul-1. This process is dependent on the COP9 signalosome Lindau protein; shRNA, short hairpin RNA. and offers a potential protective mechanism against hypoxia- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 induced proinflammatory stimuli (26). The discovery of SENP8, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202041 The Journal of Immunology 393 and in vivo. We identify a previously unappreciated role for SENP8 as a central regulator of the inflammatory process. Furthermore, we show that under physiological conditions, SENP8 functions to make mature Nedd8 available for conjugation to Culs, impacting on two transcription factors (i.e., NF-kB and HIF) important for balancing the inflammatory response. Materials and Methods Cell culture Human microvascular endothelial cell (HMEC)-1 cells were a gift of Francisco Candal (Centers for Disease Control, Atlanta, GA). HMEC-1 cells were cultured in molecular, cellular, and developmental biology- 131 medium supplemented with heat-inactivated FBS, penicillin, strepto- mycin, L-glutamine, epidermal growth factor, and hydrocortisone, as de- scribed previously (32). Stable knockdown of SENP8 was performed using FIGURE 1. Influence of LPS and adenosine on Cul-1 neddylation status. short hairpin RNA (shRNA) with the following sequences: 59-CCG- (A) LPS stimulation (10 mg/ml medium) of HMECs increases neddylation GCCTAACTTCATTCAAGACCTACTCGAGTAGGTCTTGAATGAAGT- of Cul-1 protein in a time-dependent manner. (B) Similar to previous TAGGTTTTTG-39 (TRCN0000073338, referred to as clone 38 in figures) findings in mice treated with hypoxic preconditioning, adenosine reduced or 59-CCGGGACTGTGGGATGTACGTGATACTCGAGTATCACGTAC- the LPS-induced Cul-1 neddylation in a dose-dependent manner. Repre- ATCCCACAGTCTTTTTG-39 (TRCN0000073342, referred to as clone 42 Downloaded from