In vivo expression profile of a H+-K+-ATPase
2-subunit promoter-reporter transgene
Wenzheng Zhang,1
Xuefeng Xia,1
Lei Zou,1
Xiangyang Xu,1
Gene D. LeSage,1 and
Bruce C. Kone1,2,3
1Internal Medicine and 2Integrative Biology and Pharmacology, and 3The Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Sciences Center at Houston, Houston, Texas 77030
Submitted 26 January 2003
; accepted in final form 3 February 2004
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ABSTRACT
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Because little is known about the molecular basis of transcriptional regulation of the murine H+-K+-ATPase
2 (HK
2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HK
2 promoter-reporter gene construct. In these mice, the region 7,264/+253 of the HK
2 5'-flanking region controls expression of the reporter gene enhanced green fluorescent protein (EGFP). Patterns of HK
2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of 10 major organs examined, EGFP immunoreactivity was detected abundantly in the kidney, and to a far lesser extent, in the brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of transgenic mice and colocalized with the cellular distribution of both endogenous HK
2 and aquaporin-2, consistent with the known expression pattern of endogenous HK
2 in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of the highest endogenous HK
2 expression. Although previous studies of steady-state mRNA levels suggested differences in HK
2 gene regulation in the kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HK
2 gene in these organs and suggest that regions outside the 5'-flanking region or other regulatory factors play a role in HK
2 expression in the distal colon.
transgenic mice; gene regulation
THE H+-K+-ATPASE
2 (HK
2) gene plays an important role in the control of body K+ balance (8, 23, 25). The H+-K+-ATPase also appears to contribute to bicarbonate absorption by the kidney (18, 20) and distal colon (17) and to the enhanced ammonium secretion in the inner medullary collecting duct (IMCD) during chronic hypokalemia (19). This gene was first cloned from rat distal colon (5), where it is abundantly expressed under physiological conditions. We (1) and others (2, 6, 10, 12) have demonstrated low-level, basal expression of HK
2 mRNA and protein in the kidney (2, 3, 10, 11, 14, 22), which is largely restricted to the medullary collecting ducts (1). Immunolocalization studies in the rat kidney demonstrated HK
2 exclusively in the majority of cells in outer medullary collecting duct (OMCD), where it appeared to be restricted to principal cells (22). The molecular basis for this selective expression has not been previously explored in vivo. Moreover, little is known about the transcriptional control of genes whose intrarenal expression is principally confined to the collecting duct.
We recently reported that the region spanning 7,264 to +253 of the murine HK
2 gene is basally active in the IMCD cell line mIMCD-3, but not the medullary thick ascending limb of Henle cell line ST-1 or NIH 3T3 fibroblasts (27). These results suggested the possibility that this genomic region directed collecting duct-specific expression of the HK
2 gene. On the basis of these in vitro studies, we postulated that this portion of the 5'-flanking region of the murine HK
2 gene conferred the normal, restricted pattern of HK
2 expression observed in vivo. To examine this hypothesis in adult mice in vivo, we developed lines of transgenic mice carrying an insertional promoter-reporter transgene containing the region 7,264/+253 of the murine HK
2 gene fused to an enhanced green fluorescent protein (EGFP) reporter gene and report here the expression profile of these mice. We demonstrate that the 7,264/+253 mouse HK
2 5'-flanking region is a functional promoter in vivo and mirrors the known basal expression pattern of HK
2 within the kidney in vivo. Surprisingly, however, the HK
2 promoter/EGFP transgene was not expressed in the distal colon, the site of its highest endogenous expression, implicating for the first time that differential expression of this gene in the kidney and colon has a transcriptional basis and suggesting that other regions of the HK
2 gene or regulatory controls govern HK
2 gene expression in this tissue.
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METHODS
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Preparation of promoter-reporter construct for transgenic model.
Plasmid pEGFP-1, which contained the reporter EGFP open reading frame and eukaryotic translation initiation signal, was digested with BglII/SalI. A 7.5-kb genomic DNA fragment containing 253-bp 5'-UTR sequence and 7.2-kb of the 5'-flanking regions of the murine HK
2 gene was obtained by PCR with plasmid pGL37.2MHK
2 DNA as a template (27). This 7.2-kb fragment (previously reported as GenBank/EMBL Data Bank accession no. AF350499) contains putative cis-acting DNA-regulatory sequences and a functional promoter based on studies in which it was transiently transfected into mIMCD-3 cells (27). This fragment was inserted into the BglII/SalI site upstream of the EGFP open reading frame to obtain plasmid construct 7,264/+253 mHK
2/EGFP (Fig. 1), placing the reporter gene under the transcriptional control of the murine HK
2 5'-flanking regions. The authenticity of the 7,264/+253 mHK
2/EGFP fragment was verified by DNA sequencing.
Generation of transgenic mice.
The construct 7,264/+253 mHK
2/EGFP was linearized by BglII/StuI digestion to remove vector sequences, subjected to agarose gel electrophoresis, and the DNA band was gel-purified with the GENECLEAN Turbo kit (BIOgene). Microinjections were performed in the Transgenic Facility at the Institute of Molecular Medicine for the Prevention of Human Diseases at The University of Texas Health Sciences Center, using standard protocols. Briefly, purified DNA (1 to 2 ng/µl) was microinjected into the male pronucleus of fertilized one-cell embryos from C57BL/6NHsd mice. Embryos surviving microinjection were reimplanted into the oviducts of pseudopregnant ICR females on the same day. Both the donor and recipient mice were obtained from Harlan (Houston, TX); 7.2-kb mHK/EGFP transgene-positive mice were defined by Southern blot analyses using HpaI-digested or BglII-NotI-digested genomic DNA from tail biopsy and a [32P]dCTP-labeled 0.76-kb NotI-SalI EGFP fragment from plasmid pEGFP1. To confirm further that the mice were transgenic and to determine the copy number of the transgene, a separate Southern blot analysis was performed using PvuII-digested tail DNA and a mouse HK
2-specific probe that is a 1.3-kb PvuII-SalI fragment isolated from 7,264/+253 mHK
2/EGFP construct (Fig. 1). This probe detects a 2,933-bp fragment from the transgene and a 1,451-bp fragment from the endogenous mouse HK
2 gene. The intensity of these two bands from the same mouse on the same blot was determined using the KODAK Image Station 1000. As the intensity of the 1,451-bp fragment represented the hybridization signal of the two copies of the endogenous HK
2 gene, the copy number of the transgene was determined as two times the ratio of the relative intensity of the transgene band to that of the endogenous HK
2 band. Three founder mice carrying the 7,264/+253 mHK
2/EGFP transgene were identified and bred with wild-type C57BL/6 mice to obtain hemizygous F1 progeny. F1 offspring were used for subsequent histological and immunoblot analyses. Transgenic mice used in this study were bred, housed, and monitored in accordance with the standards set by the Animal Care and Use Committee at the University of Texas Health Science Center at Houston.
Generation of antisera directed against murine HK
2.
A synthetic peptide CVELADQKDDKKFKGGKNKD, corresponding to amino acids 18 to 40 of the mouse HK
2 with a NH2-terminal cysteine residue for linkage to KLH, was used to generate anti-peptide antisera in rabbits (SigmaGenosys, The Woodlands, TX). This amino acid sequence shares <65% identity with other members of the X+-K+-ATPase gene family. The specificity of the antisera was tested by immunofluorescence microscopy and immunoblots of distal colon in the presence and absence of an excess of the immunizing peptide (see results and Fig. 2).
Fluorescence and immunofluorescence microscopy.
Transgene-positive mice or negative littermates were anesthetized with ethyl ether and perfused via the abdominal aorta with 4% paraformaldehyde in PBS. Tissues, including kidneys, distal colon, small intestine, stomach, proximal colon, heart, skeletal muscle, brain, spleen, and lungs, were isolated and immersed overnight in the same fixative at 4°C. The tissues were immersed in 20% sucrose in PBS at 4°C and then frozen in liquid nitrogen. Frozen 4- or 10-µm sections were cut with a cryostat. The sections of the same tissue from a transgene-positive mouse or a negative littermate were placed onto the same slide and frozen at 80°C until use.
Kidney tissues for histology were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and cut into 5-µm sections. Paraffin sections prepared as described above were used for fluorescence and indirect immunofluorescence microscopy. Briefly, paraffin sections of kidney were pretreated with a microwave oven for antigen recovery and then blocked with 1% normal sheep serum in 1x antibody dilution buffer (1% BSA in 0.3% Tween/PBS) for 1 h. The sections were incubated with 1:100 dilution of anti-HK
2 antiserum or polyclonal antibody directed against aquaporin-2 (from Dr. M. Knepper, National Institutes of Health) overnight at 4°C in a humidified chamber. After being washed three times in 0.3% Tween/PBS for 10 min, slides were reprobed with Alexa Fluor 594 goat anti-rabbit IgG (1:1,000; Molecular Probes, Eugene, OR) for 2 h at room temperature in a dark humid chamber and then washed in 0.3% Tween/PBS four times for 10 min/wash and rinsed in deionized water. Slides were mounted with Gel/Mount (Biomeda) and visualized. Samples incubated with preimmune serum or without primary antibody were used as negative controls. Images were acquired with a Nikon TE2000-U microscope outfitted with epifluorescence and a Cascade digital camera and processed using MetaMorph imaging and Adobe Photoshop software.
For analysis of sections from distal colon, frozen sections were warmed at room temperature for 15 min, rehydrated in PBS for 20 min, blocked in Blocking Solution 1 (5% normal goat serum, 0.3% Triton X-100 in PBS) for 30 min at room temperature, and immunolabeled with the rabbit anti-serum against mouse HK
2 (1:50 dilution) for 1 h at room temperature. As controls, sections were incubated with PBS or rabbit nonimmune serum instead of the primary antibody. After three washes in PBS, a secondary Cy5-conjugated goat anti-rabbit IgG (Amersham; 1:200 dilution) was applied for 40 min at room temperature. After final washes in PBS, the sections were mounted with an antifade reagent (Molecular Probes). The sections were then imaged using an Olympus IX71 inverted epifluorescence microscope. Data sets were acquired using a mercury short arc lamp and stored in digital format using a cooled charge-coupled device camera (Spot Insight, Diagnostic Instruments, Sterling Heights, MI).
Immunoprecipitation and Western blot analysis.
For Western analysis of EGFP expression, mouse tissue homogenates of kidney, distal colon, proximal colon, stomach, small intestine, lung, skeleton muscle, heart, spleen, and brain isolated from transgenic or wild-type mice were electrophoresed on 415% Tris·HCl Ready Gels (Bio-Rad) with SDS-PAGE running buffer and transferred by electroblotting onto nitrocellulose membranes. Blots were incubated with BD Living Colors A.V. Monoclonal Antibody JL-8 (1:1,000 dilution; Clontech) to detect EGFP signal and subsequently with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (1:5,000 dilution; Amersham). Signal detection was facilitated with enhanced chemiluminescence (Amersham). For Western blot analysis to confirm the specificity of the newly generated HK
2 antibody, mouse distal colon homogenate was used and probed with the rabbit anti-mouse HK
2 antibody in the presence or absence of excess immunizing peptide. HK
2 was immunoprecipitated from mouse kidney homogenates using the anti-HK
2 antiserum (or preimmune serum as a negative control), immobilized on protein A/G-agarose (Santa Cruz Biotechnology), washed, and eluted in Laemmli sample buffer as detailed in our earlier work (26). Immunoprecipitates were then analyzed by SDS-PAGE.
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RESULTS
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Generation of antiserum directed against murine HK
2.
We generated a polyclonal IgG specific for mouse HK
2 and tested it on immunoblots and tissue sections of mouse kidney and distal colon. The antibody specifically labeled two protein bands, a weaker band at
104 and a strong band at
110 kDa, on immunoblots of protein extracts of distal colon (Fig. 2A). These proteins were not evident when an excess of the immunizing peptide was included in the binding mixture (Fig. 2A), and the antibody did not label the basolateral membrane of the surface epithelial cells where the Na+-K+-ATPase resides (see below and Fig. 4B). Immunoblot analysis was not sufficiently sensitive to detect HK
2 in whole kidney homogenates (data not shown), presumably because the protein is expressed in only a minority of renal cells. Accordingly, immunoprecipitation experiments with the HK
2 antisera were performed. As shown in Fig. 2B, immunoprecipitation with the HK
2 antisera also revealed the
104- and
110-kDa proteins from the mouse kidney. In contrast, immunoprecipitation with preimmune serum failed to detect these proteins (Fig. 2B).
Identification of transgenic mice.
We analyzed the functional promoter activity of the murine HK
2 5'-flanking region using an insertional transgenic approach in mice. Three founders capable of germline transmission were procured. They were identified to carry the transgene 7,264/+253 mHK
2/EGFP by Southern blot analysis, using diagnostic restriction enzyme digestions. A representative Southern blot analysis using HpaI-digested tail DNA is shown in Fig. 3. As depicted in Fig. 1, HpaI cut the transgene twice with one site only 171 bp away from the 5'-end (BglII site) and the other further downstream of the EGFP coding region, producing a 8.2-kb fragment that can be detected by the EGFP-specific probe. An 8.4-kb fragment was expected from HpaI-digested genomic DNA from the corresponding region of the HK
2 gene, which cannot be detected by the EGFP-specific probe. Plasmid DNA of construct 7,264/+253 mHK
2/EGFP digested alone or digested as a mixture with tail DNA of a wild-type C57BL/6 mouse was included as a positive control. As shown in Fig. 3, a single band of
8.2 kb was present in mice 9, 10, and 14 but absent in all other mice. The bands from mice 10 and 14 migrated at the identical position as those from the positive controls. The band from mouse 9 migrated somewhat faster than those from mice 10 and 14, perhaps related to loading differences. These three female founders carrying the transgene were further confirmed by Southern blot analysis using either BglII-NotI or PvuII digestions and the same EGFP-specific or HK
2-specific probes (data not shown). No difference in band sizes among these three mice was detected in these Southern blot analyses. These results indicate that mice 9, 10, and 14 carry the full-length 7,264/+253 mHK
2/EGFP transgene. The copy numbers of the transgene in transgenic mice 9, 10, and 14 were determined as detailed in methods and found to be about 6, 8, and 8, respectively (data not shown).
F1 transgenic mice from mating of the three female founders with wild-type C57BL/6 males were screened by Southern blot analysis in the same way and used for organ harvesting. The 7,264/+253 mHK
2/EGFP transgenic mice exhibited Mendelian inheritance in these lines as expected for a single autosomal integration event. Gross anatomic examination and histological examination of major organs, including the brain, skeletal muscle, heart, kidney, small intestine, proximal and distal colon, spleen, lung, and stomach, revealed that these were phenotypically normal.
In vivo expression profile of the 7,264/+253 MHK
2/EGFP transgene.
To determine the pattern of expression of the 7,264/+253 mHK
2/EGFP transgene, brain, skeletal muscle, heart, kidney, proximal and distal colon, small intestine, spleen, and liver were harvested from multiple positive F1 mice from three founders along with their negative littermates, and tissue extracts were examined for immunoblot detection of EGFP. Abundant EGFP immunoreactivity was detected in the kidney, with much lower amounts detected in the brain and lung (Fig. 4A). Surprisingly, neither EGFP immunoreactivity (Fig. 4A) nor fluorescence (Fig. 4B) was detected in the distal colon, despite high endogenous expression of HK
2 protein in distal colon extracts (Fig. 2A) and in the apical membrane of surface epithelial cells of the distal colon by immunofluorescence microscopy (Fig. 4B). HK
2-specific immunofluorescence was notably absent from the basolateral membrane of the distal colon, indicating that there was no cross-reactivity of the anti-mouse HK
2 antisera with the Na+-K+-ATPase
1-subunit. Specific EGFP fluorescence signal was detected only in OMCD and IMCD (Fig. 5A) and colocalized with expression of endogenous HK
2 protein in these segments (Fig. 5B). The EGFP transgene was expressed in the majority of cells in these segments and colocalized with expression of aquaporin-2 (Fig. 5C), indicating expression in principal cells and consistent with previous reports of HK
2 immunoreactivity in this cell type in kidneys of normal rats (22). Expression was uniform across founders. Vascular cells, the macula densa segment, and interstitial cells within the kidney sections were negative for EGFP fluorescence. There was no significant background EGFP fluorescence in transgene-negative collecting duct nor in any of the other organs and tissues examined (not shown). Taken together, these results indicate that the 7,264/+253 region of the murine HK
2 5'-flanking region directs transgene expression primarily within the kidney that is collecting duct and apparently principal cell specific and that this promoter may contribute to HK
2 gene expression in this important cell type.
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DISCUSSION
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The in vivo functional activity of the chromatin-based 7,264/+253 nucleotide murine HK
2 transgene identifies this genomic span as a functional promoter. The proximal portion of the HK
2 5'-flanking region contains a TATA-like sequence (ATTTAA) and a CCAAT box beginning 46 and 152 bp, respectively, 5' to the transcription start site, as well as multiple binding sites for ubiquitous transcription factors such as Sp1, AP-1, NF-
B, CREB, GATA-1 (27). However, the tissue-/cell type-selective expression pattern of this promoter implicates the presence of a regulatory mechanism(s) that governs transcriptional activation of this gene in the OMCD but not other nephron segments. Our results are consistent with this conclusion and with a previous immunolocalization report (22) that, within the kidney, HK
2 appears to be largely a principal cell-specific protein.
We were surprised to find no expression of the HK
2 promoter transgene in the distal colon, the most prominent site of endogenous HK
2 expression in vivo. There are several potential explanations for this result. It is well recognized that promoter elements can reside throughout the gene, including introns and 3'-regions; thus promoter elements that direct HK
2 expression in the distal colon may reside outside of the 7.2-kb fragment we used. For example, preliminary work in our laboratory revealed a potential DNAse I hypersensitivity site specific for distal colon within intron 1 of this gene. The current results could also have been affected by not having the promoter-reporter gene constructs integrated at the native site in the genome, by transgene copy number, by secondary gene mutation unrelated to the transgene (which is estimated to occur at a frequency of 510%) (16), or by position effects related to the influence of neighboring chromosomal elements and disrupted relationships of the transgene with locus control regions and boundary elements of the chromosome (7, 15). It is possible that paracrine, autocrine, or endocrine mediators are necessary for transcriptional competency of the HK
2 promoter in the in vivo distal colon. In addition to the linear order of cis-regulatory elements in the promoter, nuclear matrix, nucleosome organization, chromatin structure, epigenetic pathways, and the ordered recruitment of transcription factors and coregulatory proteins all contribute to the accurate expression of a gene (4) and may be contributing to the lack of transgene expression in the distal colon. Studies underway in our laboratory should delineate the specific molecular mechanisms underlying this result. The fact that transgene expression was detected at low levels in the brain and lung by immunoblot analysis of EGFP expression indicates that the mechanism for silencing of the HK
2 gene in the distal colon may be specific to that tissue. Furthermore, the finding of HK
2 transgene expression in the brain and lung was not surprising, because HK
2 mRNA expression has been reported in the brain from the mouse and rabbit, and the homologous ATP1AL1 mRNA has been detected in the human brain (21). A role of HK
2 in the brain or lung function has not been established.
We observed two protein bands in HK
2 immunoprecipitates from the kidney, where only a single protein band was observed on immunoblots from the distal colon. The reason for the discrepancy is unclear but could represent posttranslational processing of HK
2 in the kidney, the expression of an alternative splice variant or of a closely related but as yet unidentified isoform. Alternative splice variants for HK
2 have been identified in the rat (9) and rabbit (2), but the mouse genomic sequence does not predict these specific variants. Further studies are planned to address this question. We also found no transgene expression in the thick ascending limb in this study in mice, although we previously reported mRNA expression in this segment in rats. Whether this discrepancy simply represents species differences, including the fact that the rat expresses alternative HK
2 mRNA variants (9), one of which (HK
2b) has not been detected as a protein in vivo, or simply mRNA expression adequate to be detected by in situ hybridization but inadequate to generate sufficient protein for detection by immunolocalization methods is unknown.
Genes whose intrarenal expression is largely restricted to the collecting duct are uncommon. A few transcripts that are selectively enriched in collecting duct cells, such as aquaporin-2, Pax-2 (13), and Hox-B7 (24), have been described. However, comparison of the 5'-flanking regions for these genes with that of HK
2 reveals little similarity in potential cis-regulatory elements. Thus this 7.2-kb segment of the 5'-flanking region of the HK
2 gene may serve as a valuable model to identify and characterize newer aspects of collecting duct-selective gene expression. This expression cassette may also serve as a powerful tool for the expression of exogenous genes, such as Cre recombinase or the Tet activator/repressor, in collecting duct cells.
In summary, we identified a 7.2-kb linear region of the murine HK
2 gene 5'-flanking region that directs specific expression of an EGFP reporter gene in the kidney, brain, and lung, but not the distal colon, with an intrarenal expression profile that mirrors that of the endogenous HK
2 protein. These results suggest that regions outside this 5'-flanking sequence direct basal HK
2 expression in the distal colon. These transgenic HK
2 promoter-reporter lines should prove to be a valuable resource for ongoing studies addressing the regulated expression of HK
2 in vivo, particularly conditions characterized by perturbations of K+ or acid-base homeostasis. Future studies incorporating the HK
2/EGFP strain of mice into other in vivo models of renal gene regulation will undoubtedly contribute pertinent information regarding the interplay of gene regulation and ion transport in the kidney and principal cells. Finally, these results provide another verifiable reason the HK
2 subunit should not be referred to as the "colonic" H+-K+-ATPase.
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GRANTS
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This work was supported by Grant R01-DK-47981 from the NIH (to B. C. Kone) and endowment funds from The James T. and Nancy B. Willerson Chair (to B. C. Kone).
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ACKNOWLEDGMENTS
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We thank Dr. M. Knepper [National Institutes of Health (NIH)] for the gift of the antibody directed against aquaporin-2 and S. Higham for expert technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and Integrative Biology and Pharmacology, The Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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