Downloaded from http://journals.lww.com/shockjournal by BhDMf5ePHKbH4TTImqenVAHxkFJp/XpPk1L/H3vMGwqMxG9jwOd8eJPG+b4DlKuAX44qu/vwzmc= on 07/30/2018 Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited. 7 EXPORTIN 1 INHIBITION ATTENUATES NUCLEAR FACTORY.BYDEPENDENT GENE EXPRESSION Mark D. Walsh Jr, Christine R. Hamiel, Anirban Banerjee, Aaron M. Cheng, Guillermo Escobar, and Robert C. McIntyre Jr Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado Received 16 Oct 2006; first review completed 6 Nov 2006; accepted in final form 18 Apr 2007 ABSTRACT—Activation of nuclear factor (NF)Y.B is mediated by signal-induced phosphorylation of I.B!, subsequent I.B! degradation, and then translocation of unbound NF-.B to the nucleus. Termination of gene expression occurs when I.B! binds NF-.B subunits (Rel A) in the nucleus. Leptomycin B specifically inhibits export of I.B! and the inactive I.B!/ Rel A complex via the nuclear export protein exportin 1. We hypothesized that inhibition of I.B! nuclear export would increase nuclear I.B! and attenuate NF-.B inflammatory gene expression in pulmonary microvascular endothelial cells. We found that inhibition of exportin 1 causes nuclear accumulation of both endogenous NF-.B (Rel A) and I.B!. IL-1" causes nuclear accumulation of NF-.B (Rel A) but does not increase nuclear I.B!. Inhibition of exportin 1 before IL-1" prevented an increase in the nuclear ratio of NF-.B (Rel A) to I.B! and decreases NF-.B DNA binding. Furthermore, inhibition of exportin 1 attenuates IL-1"Yinduced phosphorylation of I.B! without affecting I.B kinase phosphorylation. Lastly, inhibition of exportin 1 attenuates monocyte chemoattractant protein, IL-8, and intercellular adhesion molecule expression in response to IL-1" stimulation. We suggest that the decrease in cell activation due to exportin 1 inhibition is a result of termination of NF-.B DNA binding due to increased concentration of I.B! in the nucleus. KEYWORDS—Leptomycin B, nuclear shuttling, I.B!, cytokine, IL-1" INTRODUCTION The nuclear factor (NF)Y.B signaling complex is composed of five related proteins: Rel A/p65, cRel, RelB, p50, and p52 (1Y3). Active regulators of transcription exist as heterodimers, predominantly Rel A/p50; however, homodimers of p50 and p52 can repress gene transcription. The DNA binding of dimeric NF-.B is regulated by an inhibitory protein family (I.B!,I.B",I.B&) (3Y5). With IL-1" stimulation, the signal is transmitted from the receptor to TRAF6, leading to activation of the I.B kinase (IKK) complex that phosphor- ylates I.B! (6, 7). After phosphorylation, I.B! is ubiquitiny- lated and rapidly degraded. With the loss of its inhibitory protein, NF-.B translocates to the nucleus to activate tran- scription of NF-.BYdependent genes. The I.B! gene is among those strongly up-regulated, and new protein synthesis of I.B! begins rapidly within the cell (8). Free I.B! is then imported into the nucleus to bind and inactivate NF-.B. The inactive complex is then exported from the nucleus by the nuclear export protein CRM1, also known as exportin 1, which recognizes a nuclear export sequence (NES) on the I.B! molecule (9Y11). I.B" does not have the same export function as I.B! (10). Basal shuttling of NF-.B and I.B! (12, 13) occurs by spontaneous dissociation of the RelA and I.B! proteins followed by independent nuclear import, reaggregation in the nucleus, and export as a complex by exportin 1. Shuttling may be necessary for basal cellular activation and maintenance gene transcription (12). Nuclear/ cytoplasmic shuttling of NF-.B and I.B! has been examined using transformed cell lines and transfection of various constructs of NF-.B and I.B! (9Y16). Nuclear export inhibition decreases NF-.BYdependent luciferase activity; however, it is unknown if leptomycin B (LMB) would inhibit NF-.BYdependent inflammatory gene transcription in a primary cell line. Leptomycin B is a specific inhibitor of exportin 1. It functions to covalently modify a key amino acid residue in the NES recognition site of the protein (17, 18). Other cellular effects of LMB have not been described. Leptomycin B has been used to examine the physiologic role of nuclear export in cell systems. Inhibition of nuclear export prevents I.B! phosphorylation and degradation (9, 15). Further nuclear retention of the NF-.B/I.B! complex results in decreased NF-.B DNA binding (9, 10, 13, 15). Interference with shuttling may represent a novel method through which the effects of signal-induced NF-.B activation could be attenu- ated. In the current study, we hypothesized that inhibition of exportin 1 will attenuate IL-1"Yinduced NF-.BYdependent gene expression in human pulmonary microvascular endothe- lial cells (HMVECs). We used the HMVEC because it plays a central role in chemokine and adhesion moleculeYdependent HMVEC-neutrophil interactions (19Y21). The purposes of the study were to examine the effect of inhibition of nuclear export on subcellular distribution of endogenous NF-.B and I.B!. To determine if nuclear export inhibition affects proximal signal transduction, we examined activation of IKK and p38 mitogen activated protein kinase. We then sought to determine if nuclear export inhibition affects the IL-1Yinduced degradation of I.B! and NF-.B DNA binding. We also examined the effects of nuclear export inhibition on expression of intercellular adhesion molecule 1 (ICAM-1), and the cytokines IL-8 and monocyte chemoattractant protein (MCP) 1, by microvascular endothelium in response to IL-1". Our results indicate that endogenous NF-.B and I.B! are predominately cytoplasmic in the basal state. Nuclear export 160 SHOCK, Vol. 29, No. 2, pp. 160Y166, 2008 Address reprint requests to Mark D. Walsh, MD, Department of Surgery, University of Colorado Health Sciences Center, 4200 E. Ninth Avenue, Box C320, Denver, CO 80262. E-mail: mark.walsh@uchsc.edu. Supported by Grants: P50 GM049222Y12A1 and T32 GM008315Y15. DOI: 10.1097/shk.0b013e3180ca9dee Copyright Ó 2008 by the Shock Society