The Ubiquitin-Like Protein FAT10 Mediates NF- B Activation

NFB is a central mediator of innate immunity and contributes to the pathogenesis of several renal diseases. FAT10 is a TNF–inducible ubiquitin-like protein with a putative role in immune response, but whether FAT10 participates in TNF–induced NFB activation is unknown. Here, using renal tubular epithelial cells (RTECs) derived from FAT10 / and FAT10 / mice, we observed that FAT10 deficiency abrogated TNF–induced NFB activation and reduced the induction of NFB–regulated genes. Despite normal IkB degradation and polyubiquitination, FAT10 deficiency impaired TNF–induced IkB degradation and nuclear translocation of p65 in RTECs, suggesting defective proteasomal degradation of polyubiquitinated IkB . In addition, FAT10 deficiency reduced the expression of the proteasomal subunit low molecular mass polypeptide 2 (LMP2). Transduction of FAT10 / RTECs with FAT10 restored LMP2 expression, TNF–induced IkB degradation, p65 nuclear translocation, and NFB activation. Furthermore, LMP2 transfection restored IkB degradation in FAT10 / RTECs. In humans, common types of chronic kidney disease associated with tubulointerstitial upregulation of FAT10. These data suggest that FAT10 mediates NFB activation and may promote tubulointerstitial inflammation in chronic kidney diseases. J Am Soc Nephrol 21: 316–326, 2010. doi: 10.1681/ASN.2009050479 NFB is a ubiquitous transcription factor that controls the expression of genes involved in immune response, apoptosis, and cell-cycle regulation. Aberrant regulation of NFB may result in inflammatory and autoimmune diseases, impair antiviral immune responses, and contribute to malignant cellular transformation.1–3 Activation of NFB, with subsequent production of cytokines, chemokines, and adhesion molecules, is an important component of the pathogenesis of several forms of renal disease, including diabetic nephropathy (DN),4,5 hypertensive nephrosclerosis (HN),6,7 IgA nephropathy (IgAN),8 membranous glomerulopathy,9 and HIV-associated nephropathy.10 –12 The canonical NFB activation pathway can be induced by a variety of stimuli, including TNF. TNF–induced NFB activation is initiated by the engagement of TNF receptor type I at the plasma membrane, with subsequent signal transduction culminating in the activation of the I B kinase complex, which in turn phosphorylates Received May 5, 2009. Accepted October 22, 2009. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Michael J. Ross, Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: 212-2410131; Fax: 212-987-0238. E-mail: [email protected] Copyright 2010 by the American Society of Nephrology BASIC RESEARCH www.jasn.org 316 ISSN : 1046-6673/2102-316 J Am Soc Nephrol 21: 316–326, 2010 I B . Phosphorylated I B is rapidly polyubiquitinated, resulting in degradation of I B by the 26S proteasome complex, liberating NFB, which then translocates to the nucleus and activates transcription of target genes.13–16 The 26S proteasome, the main protease in eukaryotic cells, recognizes and degrades polyubiquitinated proteins.17,18 The proteolytic core complex of the 26S proteasome is the 20S core particle, which is composed of several subunits. Low molecular mass polypeptide 2 (LMP2) is an IFN–inducible subunit that can replace the constitutive subunit Y (also known as delta or 1) in the 20S particle.19 The presence of LMP2 in the 20S particle results in increased chymotryptic and tryptic activities in vitro and modulates the cleavage site preferences of the proteasome.20,21 Several studies have shown that LMP2 plays an essential role in the degradation of I B and subsequent activation of NFB.22–24 We previously reported that FAT10 is upregulated in HIVinfected renal tubular epithelial cells (RTECs) in vitro and in kidney specimens from patients with HIV-associated nephropathy and autosomal polycystic kidney disease and that increased expression of FAT10 induces apoptosis in RTECs.25 Knockout of FAT10 causes minimal phenotypic changes in unstressed mice; however, these mice exhibit increased sensitivity to death after LPS injection.26 FAT10 is constitutively expressed in mature dendritic cells and B cells27,28 and is also inducible by the proinflammatory cytokines IFNand TNFin cells of various tissue origins29,30; however, the role of FAT10 in the regulation of immune response has not been studied. Our observations that FAT10 is upregulated by HIV-1 infection of RTECs and that the pattern of FAT10 expression was similar to several other NFB–regulated genes,31 coupled with the knowledge that NFB activation is controlled at multiple levels by the ubiquitin-proteasome system, led us to test the hypothesis that FAT10 participates in the regulation of NFB activation.