microtubule, which further regulates intracellular cargo trans- port.[17,18,19] Histone deacetylase 6 (HDAC6) is associated with the microtubule network and has been shown to regulate intracellular transport of EGFR containing vesicles in some cell types through its tubulin deacetylase activity.[16,20] In HDAC6- deficient cells, the entire microtubule network becomes hyper- acetylated.[19,21,22] Whether HDAC6 regulates EGFR endocy- tic trafficking and degradation through the microtubule mediated vesicular network in cystic epithelial cells is the subject of this study. In this study, we present for the first time evidence to support the theory that HDAC6 regulates EGFR endocytic trafficking and degradation through modulation of tubulin acetylation in cystic renal epithelial cells. We found that the expression and activity of HDAC6 was upregulated in Pkd1 mutant renal epithelial cells. We further found that the stability of microtubules affected the reactive pattern of EGFR level after EGF stimulation in Pkd1 mutant kidney epithelial cells. Furthermore, HDAC6 inhibition leads to the stimulation of microtubule-dependent transport of EGFR containing vesicles and the degradation of EGFR, and normalizes EGFR localization from apical to basolateral. Results HDAC6 Expression is Increased in Pkd1 Mutant Kidney Epithelial Cells Recent evidence suggest that sorting and transport of early endocytic cargo to later stage endocytic compartments require microtubules, and HDAC6 may be involved in this process by regulating the stability of microtubules through deacetylating a- tubulin.[16,20] We found that HDAC6 was up-regulated in DBA (dolichos biflorus agglutinin) positive Pkd1 mutant mouse embry- onic kidney (MEK) epithelial cells compared to age-matched Pkd1 wild type MEK cells. HDAC6 expression was also increased in proximal tubule cells derived from postnatal day 24 (PN24) Pkd1 homozygous (Null) kidneys compared to proximal tubule cells derived from postnatal Pkd1 heterozygous kidneys (PH2) (Fig. 1A). In addition, we found that HDAC6 activity was significantly increased in Pkd1 mutant cystic epithelial cells compared with Pkd1 wild type and heterozygous control cells (Fig. 1B). HDAC6 expression was also increased in kidney tissues from well characterized, hypomorphic homozygous Pkd1nl/nl mice [23] harvested at postnatal days 7, 14 and 21, when compared to that in age matched Pkd1 wild type mouse kidneys (Fig. 1C). Our immunohistochemistry results further supported that HDAC6 was upregulated in cyst lining epithelia of Pkd1nl/nl mouse kidneys harvest at postnatal day 28 compared to that in the normal renal tubules of the age matched Pkd1 wild type mouse kidneys and the upregulated HDAC6 was localized in the cytosol, the site where HDAC6 was found in all the other cell types reported, of cystic epithelial cells (Fig. 1D). These results demonstrate an inverse relationship between PC1 expression and HDAC6 expression levels in renal epithelial cells. Inhibition of HDAC6 Activity with Tubacin, a Specific HDAC6 Inhibitor, or TSA, a Pan HDAC Inhibitor, Decreased the Expression of EGFR in Renal Epithelial Cells EGFR has been found over-expressed and hyper-activated in human ADPKD epithelia and in mouse models of ADPKD.[3] To investigate whether the upregulated HDAC6 affected EGFR levels in kidney epithelial cells, we inhibited HDAC6 activity with either tubacin or TSA. We found that treatment with either tubacin or HDAC6 Regulates EGFR Trafficking TSA decreased the levels of EGFR but increased the acetylation of a-tubulin in Pkd1 wild type and mutant kidney epithelial cells compared to untreated control cells (Fig. 2A–F). It is interesting to note that TSA treatment decreased the levels of EGFR in both Pkd1 wild type and mutant MEK cells while tubacin treatment only decease the levels of EGFR in Pkd1 mutant MEK cells (Fig. 2A and 2B). In addition, we found that TSA treatment significantly increased the acetyl-a-tubulin staining in Pkd1 wild type and mutant MEK cells as well as in PH2 and PN24 cells (data not show). Furthermore, knockdown of HDAC6 with pGIPZ lentiviral vector mediated HDAC6 shRNA in both Pkd1 wild type and mutant MEK cells decreased the expression of EGFR and increased the acetylation of a-tubulin in MEK cells compared to the control MEK cells transduced with the empty vector (Fig. 3A and 3B). In addition, we found that knockdown of HDAC6 with shRNA did not reduce EGFR expression at mRNA level in Pkd1 cells (Fig. 3C). These results suggested that HDAC6 might regulate the degradation of EGFR through deacetylation of a- tubulin. HDAC6 Associated with the Endosomal Compartments in Kidney Epithelial Cells To investigate the connection between HDAC6 and EGFR endocytic trafficking in renal epithelial cells, we first examined the association of HDAC6 with endocytic EGFR. Murine inner medullary collecting duct (mIMCD3) cells were stimulated with EGF for 10 minutes, and EGFR labeled with EGF-Alexa Fluor 488 co-localized on the early endosomes labeled with EEA1, an early endosome marker (Fig. 4A). At the same time, HDAC6 also colocalized with a significant portion of early endosomes labeled by EEA1 (Fig. 4B) and EGF-Alexa Fluor 488, respectively (Fig. 4C). These results suggest that HDAC6 interacts with the early endosomal compartment and endocytic EGFR. After cells were stimulated with EGF for 60 minutes, both HDAC6 and EGFR were colocalized with later endosomes marked by LAMP2 (Fig. 4D–4F). Colocalization of HDAC6 with EGFR as well as early and later endosomes was also confirmed in postnatal Pkd1 homozygous PN24 cells stimulated with EGF for 10 and 60 minutes (Fig. 5A–5F). In addition, we found that without EGF stimulation EGFR was randomly distributed in Pkd1 wild type and mutant MEK cells as well as in postnatal Pkd1 heterozygous PH2 and homozygous PN24 cells stained with anti-EGFR antibody (Fig. 6A). However, after stimulation with EGF for 30 min, EGFR accumulated around microtubule bundles in renal epithelial cells as shown by labeled a-tubulin staining (Fig. 6B) and this distribution pattern disappeared with EGF treatment for 60 minutes (data not shown). Inhibition of HDAC Activity Increased the Transport of the Activated EGFR from the Early Endosomes to Late Endosomes/Lysosomes We investigated whether HDAC6 affected EGFR transport from early endosomes to late endosomes/lysosomes by inhibiting HDAC6 activity with TSA. We found that although the degree of colocalization of EGFR with early endosome marker EEA1 decreased with time in mIMCD3 and PN24 cells stimulated with EGF alone (Fig. 7A and 7B), the percentage of vesicles which were positive for both EGFR and EEA1 decreased more quickly in these cells after cotreatment with EGF and TSA (Fig. 7A and 7B). We further found that more EGFR was co-localized with the late endosome and lysosome marker LAMP2 in mIMCD3 and PN24 cells upon co-treatment with EGF and TSA at later time points (Fig. 7C and 7D). These findings suggested that inhibition of PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49418