Alterations in transporter expression in liver, kidney, and duodenum after targeted disruption of the transcription factor HNF1α☆
Introduction
Transporters serve a multitude of functions, including import and export of critical endogenous substrates, as well as aiding in absorption, distribution, and elimination of many xenobiotics. Transporters characteristically move water-soluble compounds and hydrophilic metabolites into and out of cells because these molecules do not pass readily through membranes. Due to a wide range of compounds that need passage through membranes, a host of transporters have evolved to manage the cellular environment. In this regard, several transporter families exist to aid in disposition of chemicals, including the organic anion transporters (Oat), organic anion transporting polypeptide transporters (Oatp), organic cation transporters (Oct), multidrug resistance protein transporters (Mdr), multidrug resistance-associated protein transporters (Mrp), the bile acid and sterolin transporter families, along with Bcrp (breast cancer resistance protein; Abcg2). Whereas this is only a fraction of the total transporters that exist, these transporter families have been implicated as being important in the uptake and elimination of xenobiotics in liver, kidney, and intestine—all organs which play a vital role in drug disposition.
HNF1α is a transcription factor that is known to regulate the basal expression of many genes, and has been referred to as a “master” transcription factor due to its control over other known nuclear receptors and transcription factors [1]. HNF1α is expressed in liver, kidney, intestine, stomach, and pancreas [2], [3], and has been implicated in the regulation of bile acid, fatty acid, and drug metabolism in vivo [4], [5], [6]. Disruption of HNF1α results in decreased expression of several cytochrome P450s including Cyp1a2, Cyp2c29, Cyp2d9, Cyp2e1, and Cyp7b1, and increased expression of Cyp2b10, Cyp3a11, Cyp4a1, Cyp7a1, Cyp8b1, and Cyp39a1 [6]. Similarly, HNF1α is believed to be critical for regulation of insulin-like growth factor 1 (IGF-1), and the resulting loss of IGF-1 expression in the absence of HNF1α gives a phenotype resembling Laron dwarfism, and non-insulin-dependent diabetes mellitus (NIDDM) [7] suggesting the possibility of compensation by other transcription factors or altered interactions with co-activator and co-repressor proteins that are central to normal expression of HNF1α target genes [8], [9].
The development of HNF1α-null mice allows for an in vivo model for examining the physiological role of HNF1α. Mutations in the HNF1α gene exist in humans and lead to maturity-onset diabetes of the young type 3 (MODY-3), and the resulting physiological consequences seem to be modeled well by the null mouse [7].
HNF1α has also been implicated in the downregulation of only a few transporters, including human organic-anion-uptake transporters OATP-C and OATP8, the human sodium-glucose transporter (SGLT1), as well as mouse Oatp4 and the sodium-phosphate transporters Npt1, 2, and 4 in mouse [10], [11], [12]. However, this probably represents only a handful of transporters that might be regulated by HNF1α, whether direct or indirect. Decreased binding of HNF1α to 5′ flanking regions of transporters such as Ntcp and Oatps may be a critical event in bile-acid homeostasis, as this may lead to impaired hepatic uptake during cholestasis [13], [14]. However, very little work has focused on whether HNF1α regulates other solute carrier transporter families (i.e. Oats, Octs, Oatps), as well as its effects on the efflux transporters (i.e. Mdrs, Mrps). Similarly, the tissue distribution of HNF1α suggests that this transcription factor may be critical in other organs besides liver, especially in kidney and in intestine. To identify HNF1α target genes in these three tissues the branched DNA assay (Genospectra; Fremont, CA) was utilized to examine expression of a large battery of transporters in both wildtype (WT) and HNF1α-null mice. This study aims to determine whether HNF1α is critical for endo- and xenobiotic transporter expression in a variety of tissues.
Section snippets
Mice
HNF1α-null mice were engineered using Cre-loxp recombination as described previously [7]. Because HNF1α-null mice are infertile, null mice were derived by cross-breeding heterozygote male and female mice. HNF1α(+/+) littermates were used as wildtype (WT) controls. Tissues from eight male HNF1α WT and eight HNF1α-null adult mice under 4 months of age were collected and snap frozen in liquid nitrogen. Tissues were stored at −80 °C until further use.
RNA isolation
Total RNA was isolated using RNAzol Bee reagent
Hnf1α disruption alters expression of the organic anion transporter (Oat) family in liver, kidney, and duodenum
Oats are solute uptake transporters known to play an important role in kidney. Oat2, which has the highest expression of Oats in liver, had markedly lower hepatic mRNA expression in Hnf1α-null mice as compared to WT mice (Fig. 1), whereas Oat1 and Oat3 remained unchanged (Table 2). Oat1, 2 and 3 expression in kidney was lower in null than WT mice; expression of Oat1 and 2 in HNF1α-null kidney was <20% than in WT mice. In duodenum, Oat3 expression was five-fold higher in Hnf1α-null mice than in
Discussion
The results of the present study demonstrate that Hnf1α controls a multitude of transporters involved in the disposition of a wide variety of substrates. These data support the concept that Hnf1α plays a role not only in the regulation of drug and bile acid metabolism, but also in the uptake and export of substrates for these enzymes.
Hnf1α serves as a central mediator for many other transcription factors, and while much of this regulation is not understood, speculation of possible methods of
Acknowledgements
The authors would like to thank Katy Allen and Drs. Matt Dieter, Hong Lu, Chuan Chen, and Susan Buist for input and discussion, as well as the Laboratory Animal Resource Center at KUMC for their excellent contributions in helping with the mice.
References (42)
- et al.
HNF-1alpha: have bile acid transport genes found their “master”?
J Hepatol
(2002) - et al.
Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas
J Biol Chem
(1994) - et al.
Hepatocyte nuclear factor 1 alpha: a key mediator of the effect of bile acids on gene expression
Hepatology
(2003) - et al.
Regulation of the liver fatty acid-binding protein gene by hepatocyte nuclear factor 1alpha (HNF1alpha). Alterations in fatty acid homeostasis in HNF1alpha-deficient mice
J Biol Chem
(2000) - et al.
Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNF1alpha)-deficient mice
Biochem Pharmacol
(2003) - et al.
Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins
J Biol Chem
(2000) - et al.
Characterization of the human OATP-C (SLC21A6) gene promoter and regulation of liver-specific OATP genes by hepatocyte nuclear factor 1 alpha
J Biol Chem
(2001) - et al.
Hepatocyte nuclear factor 1 alpha controls renal expression of the Npt1-Npt4 anionic transporter locus
J Mol Biol
(2002) - et al.
Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis
Hepatology
(2003) - et al.
Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis
J Biol Chem
(2003)
Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter
J Biol Chem
Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver
Hepatology
Full-length cDNA cloning and genomic organization of the mouse liver-specific organic anion transporter-1 (lst-1)
Biochem Biophys Res Commun
The multidrug resistance protein family
Biochim Biophys Acta
HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF
Genes Dev
Laron dwarfism and non-insulin-dependent diabetes mellitus in the Hnf-1alpha knockout mouse
Mol Cell Biol
Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms
Endocrinology
Regulation of the human Na(+)-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1
Am J Physiol Gastrointest Liver Physiol
Endotoxin downregulates rat hepatic Ntcp gene expression via decreased activity of critical transcription factors
J Clin Invest
Tissue distribution and ontogeny of mouse organic anion transporting polypeptides (Oatps)
Drug Metab Dispos
Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism
Diabetes
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2021, Pharmacology and TherapeuticsCitation Excerpt :Several transcription factors have been identified to be involved in the regulations of OATs. For example, in hepatocyte nuclear factor 1α (HNF1α)-null mice, the levels of renal Oat1, Oat3, and Urat1 mRNA were markedly reduced as compared to those in wild-type mice, and HNF1α overexpression enhanced OAT1, OAT3, and URAT1 promoter activity in vitro (Kikuchi et al., 2006; Kikuchi et al., 2007; Maher et al., 2006; Saji et al., 2008). In ex vivo experiments with kidney organ culture, HNF4α antagonist attenuated the expression of Oat1 and Oat3 mRNA (Martovetsky, Tee, & Nigam, 2013).
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2019, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :So, it is plausible that Hnf4α mediates the effect of glucocorticoids on some multi-specific SLC transporters. In contrast, the arrhythmic expression of Hnf1α encoding the transcriptional regulator of intestinal Sglt1 (Rhoads et al., 1998; Vayro et al., 2001) and several Oat and Oatp transporters (Maher et al., 2006), excludes the possibility that this transcription factor might be a mediator of circadian- and glucocorticoid-regulated transcription of Sglt1 and Oatp1. Our study illustrates that the regulation of genes encoding SLC and ABC transporters can be interpreted in the framework of several regulatory pathways – circadian control (Mohawk et al., 2012; Richards and Gumz, 2012), neuroendocrine control of biogenesis and secretion of corticosteroids (Kalsbeek et al., 2012) and remote sensing and signaling model (Nigam et al., 2015; Wu et al., 2011).
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National Institute of Health grants ES-09716 and ES-07079.