Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells

Abstract

Cholesterol-based membrane microdomains, or lipid rafts, are believed to play important, yet poorly defined, roles in protein trafficking and signal transduction. In polarized epithelial cells, the current view is that rafts are involved in apical but not in basolateral protein transport from the trans-Golgi network (TGN). We report here that cholesterol is required in a post-TGN mechanism of basolateral regionalization. Permanently transfected Madin-Darby canine kidney cells segregated the caveolae/raft-associated high-density lipoprotein scavenger receptor class B type I (SR-BI) predominantly to the basolateral domain where it was constitutively internalized and recycled basolaterally. Acute cholesterol depletion did not significantly alter SR-BI internalization, implying a cholesterol depletion-insensitive endocytic process but instead induced its transcytosis through a protein kinase A (PKA)- and microtubule-dependent mechanism. Forskolin also elicited SR-BI transcytosis. The basolateral distribution of endogenous epidermal growth factor receptor remained unaffected. Strikingly, cholesterol depletion induced PKA activity without increasing the cAMP levels. Thus, our results are consistent with a scenario in which cholesterol-based rafts promote internalization and basolateral recycling of internalized SR-BI whereas a PKA pool sensitive to cholesterol depletion mediates SR-BI transcytosis. Regulated transcytosis of SR-BI may provide an additional mechanism to control cholesterol homeostasis. These results disclose relationships between cholesterol-based rafts and PKA activity operating in a post-TGN mechanism of regulated apical-to-basolateral cell surface protein distribution

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

“…It was recently reported that membrane cholesterol content regulates PKA activity (Burgos et al, 2004) and that the catalytic subunit of PKA was associated with cell surface caveolae (Heijnen et al, 2004). In our study, we observed colocalization of caveolin-1 and PKA in rat aortic endothelium prevented after cholesterol depletion with methyl-␤-cyclodextrin.…”

Scavenger receptor class B type I mediates biliary cholesterol secretion independent of ATP-binding cassette transporter g5/g8 in mice

“…34 In addition, a transcytotic route for SR-BI has been characterized, which mediates the movement of the receptor from basolateral to apical membranes in a microtubule-dependent fashion. 35,36 Although transcytosis of SR-BI was not formally investigated in our study, our data indicate that transcytotic pathways do not contribute to the SR-BI-mediated increase in biliary cholesterol secretion, because cholesterol output into bile was not reduced in response to colchicine, which blocks microtubule function.…”

Cholesterol-sensitive Modulation of Transcytosis

“…We also provide initial evidence suggesting that the efficiency of transcytosis is fine-tuned by changes in plasma membrane cholesterol levels. Acute cholesterol depletion may have a universal impact on the stimulation of transcytosis, exemplified here for pIgR and hTfnR, and previously for the raft-Cav1-associated (Graf et al, 1999) scavenger receptor class B type I in MDCK cells (Burgos et al, 2004). Acute cholesterol enrichment yields the opposite effect.…”

“…Transcytosis of the empty TfnR was monitored essentially as described (Burgos et al, 2004). PTR or parental MDCK cells grown on 24-mm filters were treated (or not) with m␤CD for 60 min; thereafter, the basolateral surface was biotinylated in the cold using the cleavable sulfo-NHS-SS-biotin (0.1-mg/ml).…”