HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells Wei Liu1,3,4, Lucy X. Fan1,3, Xia Zhou1,3, William E. Sweeney, Jr.1,3, Ellis D. Avner1,2,3, Xiaogang Li1,2,3* 1 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America, 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America, 3 Children’s Research Institute, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America, 4 Department of Nephrology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China Abstract We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated a-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of a-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around a-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating a-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease. Citation: Liu W, Fan LX, Zhou X, Sweeney WE Jr, Avner ED, et al. (2012) HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells. PLoS ONE 7(11): e49418. doi:10.1371/journal.pone.0049418 Editor: Bin He, Baylor College of Medicine, United States of America Received June 13, 2012; Accepted October 7, 2012; Published November 13, 2012 Copyright: 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: X. Li acknowledgments support from the Polycystic Kidney Disease Research and Education Foundation grant, Children’s Research Institute grant and National Institutes of Health grant R01DK084097. E.D. Avner acknowledgments support from National Institutes of Health grant P50DK079306 (RCEPN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: E-mail: xxli@mcw.edu Introduction Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease caused by mutations in either PKD1 or PKD2 and is characterized by the formation and progressive growth of cystic lesions that ultimately destroy normal renal parenchyma.[1,2,3] Cyst growth is the result of sustained proliferation of incomplete or de-differentiated epithelial cells and accumulation of fluid within the cysts.[3]. ErbB receptor tyrosine kinases (EGF receptor or EGFR, ErbB2, ErbB3, and ErbB4) and their ligands play important roles in renal development, in renal electrolyte homeostasis and tubule repair following injury.[4,5,6,7,8,9,10] EGFR is normally sorted to basolateral membranes in mature tubular epithelial cells.[8] However, numerous primary PKD causing mutations alter EGFR polarity, leading to increased apical expression and activity.[11] Cystic epithelial cells from ADPKD patients are unusually susceptible to the proliferative stimulus of EGF. EGF and EGF- like ligands are secreted into the apical medium of cultured cystic epithelial cells and are present in cyst fluid from ADPKD patients.[12,13] Thus, in cystic epithelia, both receptors (ErbB1 and ErbB2) and ligands are expressed on the same side of the cell leading to sustained mitogenic signaling. In addition, increased expression of ErbB2 leads to the formation of ErbB1/ErbB2 heterodimers that also slows internalization and receptor degra- dation.[14]. Inhibition of EGFR tyrosine kinase activity either genetically or pharmacologically significantly reduces renal cyst formation and improves renal function in rodent models of PKD.[7,11,15] These observations suggest that persistent EGF signaling from the apical cell surface of renal epithelia is a primary disease progression factor in PKD. However, the mechanism(s) involved in EGF mediated EGFR stability and endocytic trafficking in cystic epithelial cells is unknown. Ligand activated EGFR complexes on the apical cell surface are internalized into apical sorting endosomes (ASE) and apical recycling endosomal (AREs) intermediates and trafficked through a series of endocytic compartments where they are either recycled or sorted for proteolytic degradation in the lysosome.[16] Aberrant regulations of these complex sorting pathways have been linked to the development and progression of PKD.[14]. Microtubules, together with the microtubule-based motor proteins, kinesin and cytoplasmic dynein, are involved in sorting and transport of early endocytic vesicles to later stage endocytic compartments.[17,18] Recent studies suggest that acetylation of a- tubulin, the component of microtubules, affects the stability of PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49418