Reduced microtubule acetylation in cystic fibrosis epithelial cells

Abstract

Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-α-tubulin (Ac-tub) content is reduced by ∼40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr-/- mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-κB activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110α is also identified as a regulator of MT acetylation.

Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells

“…To test this hypothesis, expression of TPPP was reduced in immortalized human tracheal 9/HTEo 2 cells. We have previously published that microtubules in CF cell tissues exhibit reduced acetylation compared with controls (16). In this article, we present data showing reduced rates of microtubule formation.…”

“…We have demonstrated that microtubule acetylation is reduced in CF cells and tissues (16). Reduced microtubule acetylation can be a marker of instability, and may suggest alterations in the balance of microtubule dynamics.…”

“…(37,38). We have previously demonstrated that microtubule acetylation is reduced in CF cells and tissues, leading to downstream signaling changes, including increased NF-kB signaling and cholesterol accumulation (16). Reduced microtubule acetylation can be a marker of microtubule instability, so we examined microtubule dynamics in cultured and primary nasal epithelial CF cells.…”

“…We have recently demonstrated that impaired endosomal transport and perinuclear cholesterol accumulation in CF cells is a consequence of decreased acetylation of a-tubulin in CF epithelial cells, the first direct report of microtubule modifications in CF (16). Restoring microtubule acetylation by inhibition of HDAC6 improves cholesterol processing and reduces inflammatory signaling profiles, showing the potential importance of microtubule regulation in CF cell signaling (16).…”

“…Our previous work demonstrated a relationship between microtubule regulation and cholesterol accumulation as a marker of intracellular transport (16). Therefore, it was hypothesized that EPAC1 activation with 8-cpt-cA should disperse accumulated cholesterol in CF cells due to restored microtubule polymerization rates.…”