HDAC6 Regulates EGFR Trafficking Figure 6. EGFR was accumulated around microtubules following EGF stimulation. (A) EGFR was randomly distributed without EGF stimulation. Four kinds of renal epithelial cells (WT, Null, PH2, PN24) were serum-starved overnight and were then treated with 0.005% saponin before staining with anti-EGFR (green) and anti-acetyl-a-tubulin (red) antibodies. (B) EGFR was accumulated around microtubules following EGF stimulation. Serum-starved renal epithelial cells were stimulation with EGF for 30 min. The cells were then processed for immunostaining as in (A). EGFR (green) was accumulated around microtubule bundles labeled by acetyl-a-tubulin staining (red). doi:10.1371/journal.pone.0049418.g006 microtubule stability leads to the recruitment of molecular motors kinesin-1 and cytoplasmic dynein to microtubules thereby stimulating anterograde and retrograde vesicle transport including EGF stimulated EGFR.[17] The increased expression of HDAC6 and mislocalization of EGFR in cystic renal epithelial cells led us to explore the relationship between HDAC6 and EGFR in cystic epithelial cells. The data presented in this study demonstrate that HDAC6 contributes to increased expression of EGFR by disrupting microtubule mediated EGFR degradation in cystic epithelial cells. Our results show that inhibition of HDAC6 activity with TSA or HDAC6 shRNA significantly decreased the levels of EGFR and also decreased the EGFR axis mediated phosphory- lation of ERK1/2 in Pkd1 mutant renal epithelial cells. These data suggest that upregulation of HDAC6 may play a role in upregulating apical EGFR stability leading to prolonged EGFR signaling that facilitates cyst formation. Previous reports from our laboratory demonstrated that in- hibition of HDAC activity with TSA prevent renal cyst formation through targeting MEF2C or Id2 signaling, respectively.[25,26] Our results that inhibition of HDAC activity with TSA could significantly normalize the mislocalized EGFR from apical to basolateral suggested a third possible mechanism for HDAC in mediating cyst development. In sum, our study identified HDAC6 as a key component of the endocytic trafficking network in renal epithelial cells. The most exciting result related to translational application is that inhibition of HDAC6 activity with its specific inhibitor, tubacin, decreased the levels of EGFR and might also normalize the mislocalized EGFR in Pkd1 mutant MEK cells. Thus, targeting HDAC6 to downregulate EGFR and also normalize EGFR localization may be a potential therapeutic approach to treat polycystic kidney disease. Concise Methods Cell Culture and Reagents IMCD3 (murine inner medullary collecting duct) cells, HEK- 293T cells were maintained at 37uC in 5% CO2 in DMEM (Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS). Dolichos biflorus agglutinin (DBA) positive Pkd1 wild type and Pkd1 cells mouse embryonic kidney (MEK) cells were maintained as previously described.[27] Pkd1 heterozygous PH2 and homozygous PN24 cells were kindly provided by Dr. Stefan Somlo (Yale University).[28] In brief, Pkd1 cell lines were derived from a single Pkd1flox/2 carrying a conditionally immortalizing, temperature and interferon-c responsive transgene (H-2Kb-tsA58; ImmortoMouse, Charles River). Pkd12/2 were produced from Pkd1flox/2 cells by transient transfection with a plasmid expressing the Cre recombinase. Passage numbers were kept below 20 for all cell lines. Cells were maintained in DMEM/F12 supplemented with 10% FBS and c-interferon (5 U/ml; Sigma-Aldrich, MO) at 33uC and 5% CO2.[28] Trichostatin A (TSA), cycloheximide, nocodazole and saponin were purchased from Sigma-Adrich. Western Blot and Immunofluorescence Western blotting was performed on whole-cell lysates as described by the manufacturer (Upstate Biotechnology, NY). The antibodies used for Western blotting and Immunofluores- cence included anti-EGFR antibody (Cell Signaling technologies, MA; Santa Cruz, CA); anti-HDAC6 antibodies (Millipore, MA); anti-EEA1 (Santa Cruz, CA); anti-LAMP2 (Developmental PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49418