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 α-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 α-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 α-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 α-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.
Highlights
We found that histone deacetylase 6 (HDAC6) was up-regulated in Dolichos biflorus agglutinin (DBA) positive Pkd1 mutant mouse embryonic kidney (MEK) epithelial cells compared to age-matched Pkd1 wild type MEK cells
HDAC6 expression was 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)
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)
Summary
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] 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 EGFlike ligands are secreted into the apical medium of cultured cystic epithelial cells and are present in cyst fluid from ADPKD patients.[12,13] in cystic epithelia, both receptors (ErbB1 and ErbB2) and ligands are expressed on the same side of the cell leading to sustained mitogenic signaling. Increased expression of ErbB2 leads to the formation of ErbB1/ErbB2 heterodimers that slows internalization and receptor degradation.[14]
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