ARTICLE |
Correspondence to: Charles D. Boyd, Lab. of Matrix Pathobiology, the Pacific Biomedical Research Center, University of Hawai'i, 1993 EastWest Road, Honolulu, HI 96822. E-mail: cbkc08901@aol.com
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Summary |
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We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6. (J Histochem Cytochem 51:887902, 2003)
Key Words: Abcc6, Mrp6, pseudoxanthoma elasticum, tissue distribution, in situ hybridization, differential polyadenylation
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Introduction |
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ATP-binding cassette (ABC) proteins transport various substrates across biological membranes in all known organisms. These transmembrane proteins typically consist of two transmembrane domains (TMDs) and two highly conserved nucleotide-binding domains (NBDs) containing Walker A and B motives and a signature C region. In most eukaryotic ABC transporters, these domains are fused together into one polypeptide (
The subfamily C, also called the CFTR/MRP subfamily for the prominent members CFTR (cystic fibrosis transmembrane conductance regulator or ABCC7) and MRP1 (multidrug resistance-associated protein or ABCC1), consists of 12 genes in humans (
A spectrum of mutations in the human ABCC6 (MRP6, MOAT-E) gene has been shown to be responsible for pseudoxanthoma elasticum (PXE) (
Mutations in several ABCC genes result in a variety of different heritable disorders. These include mutations in ABCC7 that result in cystic fibrosis (reviewed in
The abundance of human ABCC6 in kidney and liver and the high levels of Abcc6 mRNA in rat liver have prompted some investigators to suggest that PXE, a disorder in which abnormal renal and/or hepatic function has not been observed, might be a metabolic disease (
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Materials and Methods |
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Animals
Eight-week-old male and female C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). In compliance with NIH regulations, animal handling and experiments using animal tissue were conducted according to an animal use protocol that was approved by the Institutional Animal Care and Use Committee of the University of Hawai'i.
Cloning of ABCC6 cDNAs from Mouse Liver
An 8-week-old male C57BL/6 mouse was sacrificed and the liver was excised and snap-frozen in liquid nitrogen. The frozen tissue was homogenized and resuspended in 1 ml RNA STAT-60 (TEL-TEST; Friendswood, TX) per 100 mg tissue. Total RNA was isolated according to the manufacturer's instructions and cDNA was prepared with Oligo(dT) primer using Superscript first-strand synthesis system for RT-PCR (Invitrogen; Carlsbad, CA). Based on the published murine Abcc6 mRNA sequence (GenBank accession number NM_018795), primers mAbcc6-ex30a (5' TGCGCTCCGTGATGGACTGT 3') and mAbcc6-3'UTRb (5' GTTGTAAAGACGAGTCGGTC 3') were designed to obtain a 586-bp cDNA (Abcc6-3'UTR). A 30-µl PCR reaction mixture contained 1.5 µl liver cDNA, 1 µM each primer, 200 mM dNTPs, 3 µl 10 x buffer and 1.5 µl AmpliTaq DNA polymerase (Applied Biosystems; Foster City, CA). PCR reaction conditions in a 3700 thermocycler (PerkinElmer; Wellesley, MA) were as follows: 3 min denaturation at 94C, 35 cycles of 30 sec at 94C, 30 sec at 52C, 1 min at 72C, followed by a 5-min terminal elongation step at 72C. PCR products were purified from a 1.2% TAE agarose gel with Quiaquick gel extraction kit (Qiagen; Hilden, Germany), cloned into pGEM-T easy (Promega; Madison, WI) to yield plasmid pGEM-Abcc6-3'UTR, and transformed into E. coli DH5. Plasmid midi preparations were prepared with Midiprep kit (Qiagen). The orientation of the insert was verified by restriction analysis and its sequence verified with BigDyeTerminator (Applied Biosystems) using an ABI310 automated sequencer (Applied Biosystems). Based on published EST data (GenBank accession numbers:
AW493424,
BF322691,
AI507076) and the published Abcc6 mRNA (GenBank accession number NM_018795), nested poly(dT) reverse primers mAbcc6-TC1b (5' TTTTTTTTTTGGAGTTGTAAA 3') and mAbcc6-TC2b (5' TTTTTTTTTTCACAGTTTCTC 3') were designed. These primers were used, in combination with primer mAbcc6-ex30a, to amplify two species of differentially polyadenylated Abcc6 cDNAs. PCR conditions and cloning of a 512-bp probe (Abcc6-3'UTR-TC2) into pGEM-T easy, were performed as described above.
Preparation of 33P-radiolabeled Antisense RNA Probes
One hundred µg of plasmids pGEM-Abcc6-3'UTR and pGEM-Abcc6-3'UTR-TC2 was linearized by digestion with SalI (NEB; Beverly, MA) and gel-purified. To obtain 33P-labeled Abcc6 antisense RNA probes with high specific activity (2 x 109 cpm/µg), in vitro transcriptions were performed with 5 µg linearized plasmid DNA, 2 µl 5 x transcription buffer, 40 U RNasin, 10 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 3 µM UTP, 6.4 µl [33P]-UTP (3000 Ci/mM, 10 mCi/ml), and 15 U T7 RNA polymerase in a 20-µl reaction using Riboprobe in vitro transcripton systems (Promega). A low specific activity (2 x 107 cpm/µg) GAPDH antisense control probe was transcribed from 5 µg of a linearized construct pTRI-GAPDH mouse (Ambion; Austin, TX) under the same conditions using only 0.05 µl [
33P]-UTP (3000 Ci/mM, 10 mCi/ml). Transcription was carried out for 1 hr at 37C, followed by digestion with 1 U DNase for 20 min at 37C. Incorporation of [33P]-UTP and the specific activities of the probes were determined and the radiolabeled RNA probes were gel-purified as described in the RPA III ribonuclease protection assay manual (Ambion). Probe sizes were 677 nucleotides for the Abcc6-3'UTR, 603 nucleotides for the Abcc6-3'UTR-TC2, and 403 nucleotides for the GAPDH antisense RNA probes. RNA length standards were synthesized in a similar in vitro transcription reaction using an RNA Century marker template set (Ambion).
RNase Protection Assay
Total RNA was isolated from various tissues of C57BL/6 mice with STAT-60 (TEL-TEST) as described above. RNase protection assays were carried out with RPA IIITM ribonuclease protection assay kit (Ambion) using a standard hybridization procedure described by the manufacturer. We used 40 µg of each total RNA preparation, 0.05 ng Abcc6-3'UTR, and 4 ng GAPDH 33P antisense RNA probes per sample and digested single-stranded RNA with RNase T1, diluted 1:20. RNase-protected fragments were separated on a 6% polyacrylamide, 8 M urea, TBE (45 mM Tris-borate, 1 mM EDTA) sequencing gel. The dried gel was exposed to a low-energy phosphorimager screen (Amersham Biosciences; Sunnyvale, CA) to visualize radiolabeled, size-separated RNA fragments.
RT-PCR on Human Leukocyte RNA
Total RNA was isolated from whole blood from two healthy individuals using the QIAamp RNA blood mini kit (Qiagen). cDNA was prepared from 0.4 µg total RNA with the Superscript first-strand synthesis system for RT-PCR (Invitrogen) using random hexamer primers. The mRNA sequence enclosed within exons 2526 of human ABCC1 was PCR-amplified during 35 cycles of a standard PCR reaction as described previously (
Preparation of DIG-labeled Sense and Antisense RNA Probes
A DIG RNA labeling kit (SP6/T7) (Roche; Mannheim, Germany) was used to prepare DIG-labeled Abcc6 RNA probes. SalI-digested plasmid pGEM-Abcc6-3'UTR was transcribed in vitro with T7 RNA polymerase to yield digoxigenin-labeled Abcc6 antisense probe. To obtain Abcc6 sense RNA probe, the same plasmid was digested with NcoI and was transcribed in vitro using SP6 RNA polymerase. To reduce the lengths of the probes to about 250 nucleotides, the RNA probes were then subjected to alkaline hydrolysis. DIG incorporation was determined using a DIG nucleic acid detection kit (Roche).
Western Blotting Analysis
Liver extracts and plasma membranes were prepared as described (
In Situ Hybridization
Male and female C57BL/6 mice were sacrificed by cervical dislocation. Tissues to be examined were obtained from 8-week-old male and female C57BL/6 mice and fixed overnight at room temperature in 4% formaldehyde in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) prepared with DEPC-treated water. Tissues were washed in PBS and dehydrated in a graded series of ethanol. Tissues were then cleared in xylenes and embedded in paraffin. Sections were cut at 5 µm, mounted on silane-coated slides, and deparaffinized by heating at 60C for 1 hr in a dry Caplin jar, followed by three changes of xylene, each for 15 min, and rehydrated through a graded series of ethanol and rinsed in DEPC-treated water. The slides were incubated for 30 min at 37C with 5 µg/ml RNase-free proteinase K dissolved in TE buffer (100 mM Tris-HCl, 50 mM EDTA, pH 8.0). Proteinase K digestion was terminated by incubation for 5 min in 0.2% glycine in PBS. Sections were postfixed for 10 min in 4% formaldehyde in PBS and washed twice with PBS for 5 min each. The slides were then transferred to 0.1 M triethanolamine-HCl, pH 8.0. Acetic anhydride was added to a final concentration of 0.5% (v/v) and incubated for 10 min with gentle stirring. Slides were rinsed for 1 min in PBS, dehydrated through a graded series of ethanol, and finally air-dried.
Sections were prehybridized for 1 hr at 37C with 150 µl hybridization solution without probe and then hybridized overnight at 70C with 50 µl hybridization solution. Hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 4 x SSC (0.6 M NaCl, 60 mM sodium citrate, pH 7.2), 1 mg/ml yeast tRNA, 1 x Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 10 mg/ml RNase-free BSA), 1 mg/ml denatured salmon sperm DNA, and 1 ng/µl digoxigenin-labeled RNA probe. During hybridization, sections were covered with autoclaved plastic Gel Bond Film cover slips (FMC BioProducts; Rockland, ME) and incubated with the hybridization solution in a moist chamber saturated with 4 x SSC. The slides were washed twice with 2 x SSC for 30 min at 50C and twice with NTE buffer (0.5 M NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0) for 5 min at 37C and then incubated for 30 min with 20 µg/ml RNase A in NTE buffer at 37C. After washing twice for 5 min each with NTE buffer at 37C, 30 min in 2 x SSC at 50C, 5 min at RT in 1 x SSC, and twice for 10 min in buffer 1 (0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5), the sections were blocked for 30 min with 2% normal sheep serum, 0.1% Triton X-100 in buffer 1 in a humid chamber. After a 2-hr incubation with antidigoxigeninAP Fab fragments (Roche) diluted 1:500 in buffer 1 with 1% normal sheep serum and 0.1% Triton X-100, the slides were washed three times with buffer 1 containing 0.1% Tween-20 and equilibrated for 10 min in buffer 2 (0.1 M NaCl, 0.1 M Tris-HCl, pH 9.5). Color detection was carried out overnight with detection buffer [200 µl buffer 2, 4 µl NBT/BCIP (DIG nucleic acid detection kit; Roche), 1 µl 1 M levamisole (Sigma; St Louis, MO), 10 µl 1 M MgCl2)]. After rinsing in buffer 3 (1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice in H2O, the sections were mounted with Mount Quick Aqueous (Research Products International; Palatine, IL).
Immunohistochemistry
Procedures for immunostaining using the unlabeled antibody peroxidaseanti-peroxidase technique (
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Results |
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Two Species of Abcc6 mRNA Detected by RNase Protection
To determine the levels of Abcc6 mRNA in different tissues, we performed an RNase protection assay (RPA). High amounts of human ABCC6 and rat Abcc6 mRNA have been detected in the liver (
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By in vitro transcription, we prepared both a radiolabeled GAPDH control probe of 403 nucleotides (that should give rise to a 316 nucleotide protected fragment) and an Abcc6 antisense RNA probe, Abcc6-3'UTR, of 677 nucleotides in length, that if hybridized with Abcc6 mRNA and digested with RNase in a RPA should theoretically result in a 586-nucleotide protected radiolabeled fragment. However, when we hybridized total RNA from mouse liver with the Abcc6 antisense probe in an RPA, in addition to the expected band at 586 bases we observed another protected fragment of about 500 nucleotides (Fig 1B, Lane 5). This band occurred consistently with different RNA preparations from liver and from other tissues (Fig 1B, Lanes 5, 6, 8, 10, and 11) and persisted under varying RNase digestion conditions (not shown). Moreover, the ratio of both protected Abcc6-3'UTR fragments was different with RNAs from different tissues. In the eyes and brain, the 586-nucleotide fragment was more abundant than the smaller RNA fragment. In the kidney, intestine and colon, the smaller RNA was slightly more abundant and, in the liver, the shorter fragment was the prominent protected species. Because Abcc6-3'UTR contains part of the mouse 3' untranslated region of the Abcc6 mRNA, a shorter protected fragment may arise as a consequence of differential polyadenylation of mouse Abcc6 mRNA. Indeed, a BLAST search of mouse ESTs with the 3'UTR probe identified several matches. Three of these matches represented mouse ESTs found in RNA isolated from the cerebellum, liver, and diaphragm (GenBank accession numbers AW493424, BF322691, AI507076) that all had a defined poly(A) sequence 100 bases upstream of the published Abcc6 mRNA sequence.
On the basis of this information, we designed nested poly(dT) reverse primers mAbcc6-TC1b and mAbcc6-TC2b (Fig 1A) and used these primers, in combination with forward primer mAbcc6-ex30a, to amplify two species of differentially polyadenylated mRNAs by RT-PCR from liver RNA. We obtained a PCR product of 600 bp with primers mAbcc6-TC1b and mAbcc6-ex30a and a 512-bp product with primers mAbcc6-TC2b and mAbcc6-ex30a (data not shown). The sequences of the two PCR products identified the two differentially polyadenylated mRNA species, as suggested by the EST data and the published mRNA.
To finally verify if the shorter band that we obtained in the RPA with the original Abcc6-3'UTR probe corresponded to an Abcc6 mRNA with a shortened 3'UTR, we cloned the shorter PCR product into the same expression vector and in vitro synthesized radiolabeled probe Abcc6-3'UTR-TC2. RPA with this probe indeed yielded a 512-nucleotide protected fragment when hybridized with liver total RNA (Fig 1C, Lane 6; the 12 extra nucleotides are 12 adenosyl residues from the nested poly(dT) primer). Taken together, these results suggest that there are two differentially polyadenylated species of Abcc6 mRNA present in different mouse tissues and that the ratio of these two mRNAs varies in different tissues.
Tissue Distribution of Abcc6 mRNAs Detected by RNase Protection
Abcc6 mRNA levels in mouse are the highest in the liver (Fig 1B, Lane 5), the amount of mRNA in the kidney is considerably lower (Fig 1B, Lane 6). Intestine and colon show similarly low amounts of Abcc6 mRNA (Fig 1B, Lanes 10 and 11), and Abcc6 mRNA levels in the mouse brain and eye (Fig 1B, Lanes 7 and 8) are even lower. There are very faint bands corresponding to extremely low levels of Abcc6 mRNA in the stomach, heart, trachea, bladder, tongue, testis, and aorta (Fig 1B, Lanes 12, 13, 15, 16, 19, 20, and 23). Abcc6 mRNA in all the other tissues included was not detectable in this RPA with 40 µg total RNA per tissue.
ISH Reveals Abcc6 Expression in Various Cell Types and in Many Organs
To visualize which cells contain Abcc6 mRNA in tissues having high to low levels of transcripts as measured by RPA and to see if we could detect Abcc6 message in some additional cells in other tissues, we performed non-radioactive ISH (
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In the liver, staining was evident in all hepatic cells but was most intense in the epithelium of the bile ducts at the portal area (Fig 2A). Kupffer cells (Fig 2A, inset) and endothelial cells (not shown) were also stained. In the kidney, staining was most intense in cells of the proximal convoluted tubules and, to a lesser degree, in cells of the distal tubules (Fig 2C). The staining in the tubules of the medulla was generally weaker. Arterial endothelial cells were also stained (not shown). In the cerebrum, most neurons were heavily stained (Fig 2E). The ependymal epithelium lining the ventricles was also strongly stained (not shown). The strong staining we observed in neurons is somewhat discordant with the results obtained in the RNase protection assay that indicated the presence of only very low amounts of Abcc6 mRNA in the brain. A possible explanation might be that these very low amounts of message are locally concentrated on the rough ER around the nuclei of the neurons, whereas the more abundant Abcc6 mRNA in hepatocytes is distributed throughout the entire cytoplasm, thus resulting in an in situ staining of similar intensity but over a much larger surface. We could also detect Abcc6 message in neurons of the spinal cord (not shown). Neurons in all layers of the retina also contained Abcc6 mRNA (Fig 2G). The corneal epithelial cells and, to a lesser degree, fibroblasts and muscle cells in the sclera of the eye were also stained (not shown).
Muscle cells in the atria of the heart were heavily stained (Fig 3A), and those in the ventricles revealed moderate staining (not shown). Abcc6 mRNA was also detected in the endothelial cells of the endocardium and in the endothelial cells and smooth muscle cells of arteries, included in the heart tissue section (not shown). Staining of the endothelial cells and smooth muscle cells was especially evident in sections of the aorta (Fig 3C). Some staining could also be observed in the fibroblasts of the intima. We also observed strong staining of leukocytes in the blood (Fig 3E) and of lymphocytes in the thymus and lymph nodes (not shown). To confirm the presence of Abcc6 mRNA in leukocytes, we isolated total RNA from human buffy coat preparations and performed RT-PCR. As is evident from Fig 5, we were able to detect ABCC6 mRNA in leukocytes from normal individuals.
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Keratinocytes in the epidermis of the skin and, to a lesser extent, fibroblasts and muscle cells in the dermis contained Abcc6 mRNA (Fig 3G). ISH revealed strong staining in the ciliated epithelium of the mucosa and in chondrocytes of the tracheal cartilage (Fig 3I). Bronchial epithelial cells were heavily stained (not shown). In the tongue, both filiform and fungiform papillal squamous epithelia were intensely stained (Fig 4A). In the stomach (Fig 4C), small intestine (Fig 4E), and colon (Fig 4G) gland cells, especially those in the basal layer of the mucosa, were strongly stained.
Although in some tissues Abcc6 mRNA levels were too low to be detected by RPA, ISH demonstrated the presence of Abcc6 mRNA in various epithelial cells known to be involved in secretion, endothelial cells, smooth and striated muscle cells, neurons in the cerebrum, spinal cord and retina, and leukocytes.
IHC Detection of Abcc6 Using the Rabbit Polyclonal Antibody HB-6
The rabbit polyclonal peptide antibody HB-6 has recently been shown to recognize the human ABCC6 protein by Western blotting analysis (
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Epithelial cells in the proximal convoluted tubules in the inner cortex of the kidney show basolateral membrane localization of Abcc6 (Fig 7A). We also observed a "dotted" staining within these epithelial cells, indicating a concentration of Abcc6 transporter molecules in some areas of the plasma membrane (Fig 7A). Epithelial cells in the distal tubules and other structures in the medulla are only moderately stained. Epithelial cells within the proximal tubules of the outer cortex and the strongly stained hepatocytes in the liver (not shown) showed predominantly cytoplasmic staining for Abcc6.
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Consistent with our results on demonstrating Abcc6 mRNA in the retina, we observed antibody staining in all neuron layers of the retina (Fig 7B). In addition to other neurons in the cerebrum (Fig 7C), Purkinje cells in the cerebellum showed strong staining with the ABCC6 antibody (Fig 7D). Cells of the ependymal epithelium were also stained (not shown). In large ganglion cells in the spinal cord, we observed staining of the Nissl bodies (not shown).
In sections of the intestine, strong immunostaining for the Abcc6 protein was seen in the surface cells of the mucosa. This observation is in contrast to the predominant staining for mRNA that we observed in the basal cells of the mucosa (Fig 7E). A similar finding was noted in the mucosa of the colon (not shown). The surface of apical cells of the squamous epithelium of the tongue was also more strongly stained with HB-6 than the more basal cell layers (not shown), again in contrast to the stronger staining that we observed by the ISH in the basal cells.
In the peripheral blood, leukocytes and the concave surfaces of erythrocytes show immunostaining with the polyclonal ABCC6 antibody (Fig 7F). Plasma membranes of some but not all lymphocytes in the lymph nodes also show Abcc6 immunostaining (Fig 7G).
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Discussion |
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In this report we have demonstrated the presence of Abcc6 mRNA and Abcc6 protein in various mouse tissues and cell types of normal mice. Tissue distribution of the human and rat orthologues of Abcc6 have previously been studied by RT-PCR (
We identified two differentially polyadenylated species of Abcc6 mRNA. The ratio of these two species varied among different tissues. The 3'UTR of Abcc6 mRNA does not contain the consensus AATAAA polyadenylation signal that is found in 90% of mammalian mRNAs, but considerable variation in polyadenylation signals has previously been reported (
This study reports the cellular localization of Abcc6 mRNA by ISH in a number of different cell types and confirms the tissue distribution by immunohistochemistry. In agreement with our data, rat Abcc6 and human ABCC6 have previously been shown to be localized to the basolateral plasma membrane in hepatocytes and kidney proximal tubules (
In tissue sections of the small intestine and colon, we observed high levels of Abcc6 mRNA in the basal gland cells of the mucosal epithelium and less staining in the surface cells. However, by IHC Abcc6 protein was more abundant in the apical cells. This difference in mRNA vs protein abundance is probably a consequence of the rapid turnover of these epithelial cells by upward migration from localized regions of cell proliferation in the crypts of Lieberkuhn (and high mRNA levels) to the mucosal surface in the colon and villi in the small intestine containing terminally differentiated epithelial cells of low proliferative capacity (
Abcc1 (Mrp1) is found in the basolateral membrane of cells of Henle's loop and in the cortical collecting duct in the kidney of mice (
Human ABCC1 (MRP1) has previously been shown to exhibit almost ubiquitous tissue distribution (
ABCC1 in humans and rats is localized basolaterally in epithelial cells of the choroid plexus in the brain, conferring a basal-to-apical drug permeation barrier, and it has been suggested that ABCC1 may contribute to the bloodcerebrospinal fluid drug permeability barrier (
An additional hypothetical function of Abcc6/ABCC6 may be in protecting not only the cytoplasm, as has been speculated for ABCC1, but also the extracellular space against oxidative stress. For example, the enzymatic activity of lysyl oxidases (amine oxidases that catalyze the crosslinking of collagen and elastin (reviewed in
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Acknowledgments |
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Supported by NIH grants EY13019 and RR16453.
We are grateful to Drs Andras Varadi and Balazs Sarkadi for insightful comments regarding the possible functions of ABCC6.
Received for publication October 10, 2002; accepted February 10, 2003.
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