Mechanisms of Altered Cholesterol Metabolism in Cystic Fibrosis

Cystic Fibrosis (CF) is a genetic disease caused by a mutation in the Cftr gene resulting in either an absent or nonfunctioning CFTR protein. CFTR is a membrane-bound chloride channel whose activation is dependent on cAMP via protein kinase A (PKA). CF cells are characterized by several phenotypes including perinuclear cholesterol accumulation and altered cell signaling profiles such as increased cAMP response element binding protein (CREB) activation. The hypothesis of this study is that the protein beta-arrestin-2 (betaarr2) is an initiating factor in propagating many of these phenotypes, specifically intracellular cholesterol transport and CREB regulation. Arrestins are multifunctional proteins that influence several cell regulatory pathways including the regulation of G-protein coupled receptors. A model cell line that overexpresses betaarr2 was developed to determine if arrestin expression recapitulates CF-like phenotypes.;Expression of pCREB normalized to total CREB (tCREB) content in betaarr2 overexpressing cells is significantly increased compared to controls, similar to what is observed in multiple CF models. Mechanistic studies reveal that betaarr2 expression leads to the activation of the Src/ERK pathway, and that stimulation of this pathway accounts for increased CREB activation in betaarr2-expressing and CF models. To test directly the impact of betaarr2 on CREB activation in CF, Cftr/betaarr2 double knockout mice were developed and analyzed for pCREB expression. Ratios of pCREB/tCREB are reduced 75% in DKO mice compared to CF mice.;betaarr2 overexpressing cells also demonstrate CF-like perinuclear localization of free cholesterol as shown by filipin stain. In order to elucidate the full effect of betaarr2 on CF-like cholesterol accumulation, two different CF model cells (pCEPR and IB3) were transiently transfected with a GFP-tagged betaarr2 shRNA construct to reduce betaarr2 expression. Both the pCEPR and IB3 cells transfected with betaarr2-GFP shRNA show reduced intracellular cholesterol accumulation. DKO mice also show normalization of cholesterol processing markers. These data support a key role for betaarr2 in modulating intracellular cholesterol transport.;Taken together, these data demonstrate that betaarr2 is a key regulator of CF-specific phenotypes and provides insight into how cells respond to the loss of CFTR function. Further understanding these mechanistic relationships will aid in the development of future therapeutics.