Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.
PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expre...
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2013-01-01
|
Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC3720616?pdf=render |
id |
doaj-0996abaaaa714aca97c1b49382bd5ac1 |
---|---|
record_format |
Article |
spelling |
doaj-0996abaaaa714aca97c1b49382bd5ac12020-11-25T02:22:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6972510.1371/journal.pone.0069725Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.Kosuke TsukamotoLorenna BuckWalker InmanLinda GriffithOlivier KocherMonty KriegerPDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms.Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo.Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.http://europepmc.org/articles/PMC3720616?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kosuke Tsukamoto Lorenna Buck Walker Inman Linda Griffith Olivier Kocher Monty Krieger |
spellingShingle |
Kosuke Tsukamoto Lorenna Buck Walker Inman Linda Griffith Olivier Kocher Monty Krieger Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. PLoS ONE |
author_facet |
Kosuke Tsukamoto Lorenna Buck Walker Inman Linda Griffith Olivier Kocher Monty Krieger |
author_sort |
Kosuke Tsukamoto |
title |
Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. |
title_short |
Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. |
title_full |
Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. |
title_fullStr |
Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. |
title_full_unstemmed |
Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1. |
title_sort |
challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic hdl receptor sr-bi regulation by its cytoplasmic adaptor pdzk1. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2013-01-01 |
description |
PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms.Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo.Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions. |
url |
http://europepmc.org/articles/PMC3720616?pdf=render |
work_keys_str_mv |
AT kosuketsukamoto challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 AT lorennabuck challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 AT walkerinman challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 AT lindagriffith challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 AT olivierkocher challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 AT montykrieger challengesinusingculturedprimaryrodenthepatocytesorcelllinestostudyhepatichdlreceptorsrbiregulationbyitscytoplasmicadaptorpdzk1 |
_version_ |
1724862888853635072 |