HDAC6 Regulates EGFR Trafficking Figure 2. Inhibition of HDAC6 activity decreased the levels of EGFR and increased acetyl-a-tubulin in renal epithelial cells. (A) and (B) Western blotting analysis of the expression of EGFR and acetyl-a-tubulin in Pkd1 wild-type (WT) MEK cells (A) and Pkd1null/null (Null) MEK cells (B) treated with or without tubacin. Tubacin (5 mM) treatment deceased the levels of EGFR in Pkd1 mutant MEK cells, but increased the acetyl-a-tubulin in both wild-type and mutant MEK cells. (C) and (D) Western blotting analysis of the expression of EGFR and acetyl-a-tubulin in Pkd1 wild-type (WT) MEK cells (C) and Pkd1null/null (Null) MEK cells (D) treated with or without TSA (100 ng/ml). TSA treatment decreased the levels of EGFR and increased the acetyl-a-tubulin in Pkd1null/null (Null) MEK cells. (E) and (F) TSA (200 ng/ml) has the same effect on the expression of EGFR and acetyl-a-tubulin in postnatal Pkd1 heterozygous PH2 and homozygous PN24 cells. * p , 0.05. ** p , 0.01. doi:10.1371/journal.pone.0049418.g002 Inhibition of HDAC Activity Decreased the Phosphorylation of ERK and Normalized EGFR Localization from Apical to Basolateral in Pkd1 Knockout Mouse Kidneys The EGFR axis is considered to act primarily through activation of the MAPK (RAS/RAF/MEK/ERK) pathway.[15] Since inhibition of HDAC6 activity increased the degradation of EGFR in renal epithelial cells, we suspected that it might also affect EGFR mediated ERK activation characterized with the levels of the phosphorylation of ERK1/2. To support this hypothesis, we found that knockdown HDAC6 with shRNA (Fig. 10A) or inhibition of HDAC6 activity with TSA (Fig. 10B) demonstrated a developmental decrease in EGFR and correlated with a substantial decreased phospho-ERK1/2 in cystic epithelial cells treated with EGF. The mislocalization of the EGFR from basolateral to apical is known to occur in kidneys of both humans and mice and is a common end point associated with several different forms of polycystic kidney disease that are initiated by mutations in different genes.[7] We provided evidence that upregulated HDAC6 regulated EGFR endocytic trafficking and degradation in cystic epithelial cells. We suspected that upregulated HDAC6 might also contribute to the mislocalization of the apical EGFR on cyst lining epithelia. To test this hypothesis, we treated Pkd1flox/ flox:Ksp-cre pups with 0.5 mg/g TSA or DMSO (control) daily from postnatal day 3 (P3) to P6 and harvested the kidneys at postnatal day 7 (P7). Immunohistochemical staining of the control DMSO treated kidneys illustrates the expression of EGFR on apical cell surfaces of cystic epithelia (Fig. 10C). In contrast, treatment with TSA significantly increased the basolateral localization of EGFR on cystic epithelia (Fig. 10D), which suggested that inhibition of HDAC activity could normalize the mislocalized EGFR from apical to basolateral. Discussion In this study, we provided the first evidence that 1) HDAC6 regulates EGFR intracellular trafficking and degradation in renal epithelial cells through HDAC6 mediated a-tubulin deacetylation and microtubule stability; 2) inhibition of HDAC activity normalizes apical EGFR to basolateral in Pkd1 conditional knockout mouse model. We found that HDAC6 was up-regulated in Pkd1 mutant MEK cells and in Pkd1 hypomorphic homozygous (Pkd1nl/nl) kidney tissues (Fig. 1A–1D). Pharmacologic and shRNA mediated inhibition of HDAC6 decreased the levels of EGFR in renal epithelial cells (Fig. 2 and 3). We further found that HDAC6 regulated EGFR intracellular trafficking and degradation along microtubules in normal and mutant renal epithelial cells by mediating microtubule acetylation and stability since 1) inhibition PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49418