Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats.
BACKGROUND
Hepatocyte nuclear factor-4α (HNF4α; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4α causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4α in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4α protein, due, in part, to the limited availability of human HNF4α-specific antibodies.


RESULTS
Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4α fusion proteins as the immunizing agent. The mouse anti-human HNF4α monoclonal antibody (K9218) generated against human HNF4α1/α2/α3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4α proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4α protein, but HEK293 showed no expression of HNF4α protein. Nuclear-specific localization of the HNF4α protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4α protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4α protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4α in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4α mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis.


CONCLUSIONS
These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4α and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4α isoforms in humans and in several other mammalian species.
Jiang
Shuying
S
Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Tanaka
Toshiya
T
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.|Pharmacology II, Department of Research and Development, Grelan Pharmaceutical Co. Ltd., Tokyo, Japan.
Iwanari
Hiroko
H
Institute of Immunology Co. Ltd., Tokyo, Japan.
Hotta
Hiromitsu
H
Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Yamashita
Hisahiko
H
Institute of Immunology Co. Ltd., Tokyo, Japan.
Kumakura
Junko
J
Institute of Immunology Co. Ltd., Tokyo, Japan.
Watanabe
Yuichiro
Y
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.|Biological Chemistry III, New Drug Research Department, Kowa Co. Ltd., Tokyo, Japan.
Uchiyama
Yasutoshi
Y
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
Aburatani
Hiroyuki
H
Genome Science Division, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
Hamakubo
Takao
T
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
Kodama
Tatsuhiko
T
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
Naito
Makoto
M
Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.