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Expression of HNF4alpha in the human and rat choroid plexus: implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier.

Niehof M, Borlak J - BMC Mol. Biol. (2009)

Bottom Line: We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.Our study evidences activity of HNF4alpha in human and rat choroid plexus.This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Toxicology and Experimental Medicine, Center of Molecular Medicine and Medical Biotechnology, Hannover, Germany. niehof@item.fraunhofer.de

ABSTRACT

Background: The choroid plexus consists of highly differentiated epithelium and functions as a barrier at the interface of the blood-cerebrospinal-fluid (CSF). This tissue may therefore determine the bioavailability and transport of drugs to the brain. Little is known about the expression of drug and xenobiotic metabolizing enzymes (DME) and of drug transporters in the human choroid plexus. Notably, the transcription factor and zinc finger protein HNF4alpha is a master regulator of DMEs and of drug transporters. As of today its activity in the blood-CSF barrier is unknown. Here we report our efforts in determining HNF4alpha activity in the regulation of ABC transporters in the human and rat choroid plexus.

Results: We report expression of HNF4alpha by qRT-PCR and by immunohistochemistry and evidence transcript expression of the ATP-binding cassette transporters ABCB1, ABCB4, ABCC1-6 in choroid plexus. Additionally, HNF4alpha DNA binding activity at regulatory sequences of ABCB4 and ABCC1 was determined by EMSA bandshift assays with a specific antibody. We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.

Conclusion: Our study evidences activity of HNF4alpha in human and rat choroid plexus. This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.

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Immunohistochemical detection of HNF4α in the choroid plexus. Slices of human (A, B) and rat (C, D) choroid plexus probes were stained with polyclonal antibodies against HNF4α. Arrows indicate representative HNF4α positive cells (A, C). To confirm specificity of the immunohistochemical localization antibodies were preabsorbed with excess of antigens for HNF4α (B, D). Patient identification numbers were indicated respectively and patient characteristics are given in Table 5. Magnification ×400.
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Figure 2: Immunohistochemical detection of HNF4α in the choroid plexus. Slices of human (A, B) and rat (C, D) choroid plexus probes were stained with polyclonal antibodies against HNF4α. Arrows indicate representative HNF4α positive cells (A, C). To confirm specificity of the immunohistochemical localization antibodies were preabsorbed with excess of antigens for HNF4α (B, D). Patient identification numbers were indicated respectively and patient characteristics are given in Table 5. Magnification ×400.

Mentions: Initially, we searched for HNF4α transcripts in individual samples of human and rat choroid plexus and confirmed gene expression of HNF4α by quantitative real time RT-PCR (Figures 1A). We found HNF4α transcript expression in human and rat choroid plexus to account for approximately a tenth of its expression in the liver (Figures 1A). It is of considerable importance that HNF4α expression in the human and rat choroid plexus is restricted to P1 promoter driven isoforms (Table 1). Furthermore, we studied expression of the insulin-like growth factor 2 (IGF2), transthyretin (TTR) and the transcription factor FOXJ1 to further qualify choroidal epithelial cells of the brain [18,19]. These transcripts are specifically enriched in choroid plexus. We observed abundant expression of IGF2, TTR and FOXJ1 in human choroid plexus as compared to total brain RNA extracts (Figures 1B). There is the need to study histological well qualified tissue, as studies with total brain RNA extracts would render findings meaningless as will be discussed later on. Unfortunately, sufficient human choroid plexus tissue suitable for the harvest of nuclear protein and to perform western blotting as well as EMSA assay could not be obtained. We nonetheless demonstrate HNF4α protein expression by immunohistochemistry by use of a specific HNF4α antibody for human (Figures 2A) and rat choroid plexus (Figures 2C). To confirm specificity an excess of antigen preabsorbed to the antibody was used (Figures 2B, D).


Expression of HNF4alpha in the human and rat choroid plexus: implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier.

Niehof M, Borlak J - BMC Mol. Biol. (2009)

Immunohistochemical detection of HNF4α in the choroid plexus. Slices of human (A, B) and rat (C, D) choroid plexus probes were stained with polyclonal antibodies against HNF4α. Arrows indicate representative HNF4α positive cells (A, C). To confirm specificity of the immunohistochemical localization antibodies were preabsorbed with excess of antigens for HNF4α (B, D). Patient identification numbers were indicated respectively and patient characteristics are given in Table 5. Magnification ×400.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713241&req=5

Figure 2: Immunohistochemical detection of HNF4α in the choroid plexus. Slices of human (A, B) and rat (C, D) choroid plexus probes were stained with polyclonal antibodies against HNF4α. Arrows indicate representative HNF4α positive cells (A, C). To confirm specificity of the immunohistochemical localization antibodies were preabsorbed with excess of antigens for HNF4α (B, D). Patient identification numbers were indicated respectively and patient characteristics are given in Table 5. Magnification ×400.
Mentions: Initially, we searched for HNF4α transcripts in individual samples of human and rat choroid plexus and confirmed gene expression of HNF4α by quantitative real time RT-PCR (Figures 1A). We found HNF4α transcript expression in human and rat choroid plexus to account for approximately a tenth of its expression in the liver (Figures 1A). It is of considerable importance that HNF4α expression in the human and rat choroid plexus is restricted to P1 promoter driven isoforms (Table 1). Furthermore, we studied expression of the insulin-like growth factor 2 (IGF2), transthyretin (TTR) and the transcription factor FOXJ1 to further qualify choroidal epithelial cells of the brain [18,19]. These transcripts are specifically enriched in choroid plexus. We observed abundant expression of IGF2, TTR and FOXJ1 in human choroid plexus as compared to total brain RNA extracts (Figures 1B). There is the need to study histological well qualified tissue, as studies with total brain RNA extracts would render findings meaningless as will be discussed later on. Unfortunately, sufficient human choroid plexus tissue suitable for the harvest of nuclear protein and to perform western blotting as well as EMSA assay could not be obtained. We nonetheless demonstrate HNF4α protein expression by immunohistochemistry by use of a specific HNF4α antibody for human (Figures 2A) and rat choroid plexus (Figures 2C). To confirm specificity an excess of antigen preabsorbed to the antibody was used (Figures 2B, D).

Bottom Line: We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.Our study evidences activity of HNF4alpha in human and rat choroid plexus.This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Toxicology and Experimental Medicine, Center of Molecular Medicine and Medical Biotechnology, Hannover, Germany. niehof@item.fraunhofer.de

ABSTRACT

Background: The choroid plexus consists of highly differentiated epithelium and functions as a barrier at the interface of the blood-cerebrospinal-fluid (CSF). This tissue may therefore determine the bioavailability and transport of drugs to the brain. Little is known about the expression of drug and xenobiotic metabolizing enzymes (DME) and of drug transporters in the human choroid plexus. Notably, the transcription factor and zinc finger protein HNF4alpha is a master regulator of DMEs and of drug transporters. As of today its activity in the blood-CSF barrier is unknown. Here we report our efforts in determining HNF4alpha activity in the regulation of ABC transporters in the human and rat choroid plexus.

Results: We report expression of HNF4alpha by qRT-PCR and by immunohistochemistry and evidence transcript expression of the ATP-binding cassette transporters ABCB1, ABCB4, ABCC1-6 in choroid plexus. Additionally, HNF4alpha DNA binding activity at regulatory sequences of ABCB4 and ABCC1 was determined by EMSA bandshift assays with a specific antibody. We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.

Conclusion: Our study evidences activity of HNF4alpha in human and rat choroid plexus. This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.

Show MeSH