ARTICLE |
Correspondence to: Juha Kere, Finnish Genome Center, PO Box 2, Tukholmankatu 2, 00014 University of Helsinki, Finland. E-mail: Juha.Kere@helsinki.fi
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutated alleles of the SLC26A2 (diastrophic dysplasia sulfate transporter or DTDST) gene cause each of the four recessive chondrodysplasias, i.e., diastrophic dysplasia (DTD), multiple epiphyseal dysplasia (MED), atelosteogenesis Type II (AO2), and achondrogenesis Type IB (ACG1B). SLC26A2 acts as an Na+-independent sulfate/chloride antiporter and belongs to the SLC26 anion transporter gene family, currently consisting of six homologous human members. Although Northern analysis has indicated some expression in all tissues studied, the only tissue known to be affected by SLC26A2 mutations is cartilage. Abundant SLC26A2 expression has previously been detected in normal human colon by in situ hybridization. We have used in situ hybridization and immunohistochemistry to examine multiple normal tissues for the expression of human SLC26A2. As expected, a strong signal for SLC26A2 mRNA and protein immunostaining were detected in developing fetal hyaline cartilage, while bronchial cartilage showed mRNA expression in adult tissues. SLC26A2 expression could also be detected in eccrine sweat glands, in bronchial glands, and in placental villi. In addition, immunoreactivity for the SLC26A2 protein was observed in exocrine pancreas. Our results suggest a more limited expression pattern for SLC26A2 than that found by Northern analysis. However, SLC26A2 expression is also detected in tissues not affected in chondrodysplasias caused by SLC26A2 mutations.
(J Histochem Cytochem 49:973982, 2001)
Key Words: DTDST, immunohistochemistry, human, expression, SLC26
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SLC26A2 (also known as diastrophic dysplasia sulfate transporter, or DTDST) is an anion transporter that is responsible for four recessively inherited chondrodysplasias of increasing severity, i.e., multiple epiphyseal dysplasia (MED;
SLC26A2 acts as an Na+-independent sulfate/chloride antiporter (
Although the phenotype caused by SLC26A2 mutations suggests cartilage as the major expression site, Northern analysis has shown wide expression patterns, with some expression in all tissues studied (
In this study we investigated multiple normal tissues to determine the expression patterns of the human SLC26A2 gene and protein. Characterization of tissues and specific cell types that express SLC26A2 in vivo is important for elucidating the physiological function of the normal protein and the pathophysiology of MED, DTD, AO2, and ACG1B.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR Analysis of Expression
PCR analyses were done using PCR-ready human MTC panel I and II cDNAs (Clontech; Palo Alto, CA). Panels included cDNA samples from brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, peripheral blood leukocyte, prostate, small intestine, spleen, testis, and thymus. Cartilage cDNA was used as a positive control. The ribs of a fetus at gestational age of approximately 20 weeks were first homogenized and total RNA was extracted using the RNeasy RNA Extraction Kit (Qiagen; Chatsworth, CA). First-strand cDNA was synthesized with TaqMan Gold RT-PCR Kit (Applied Biosystems; Foster City, CA) from 0.4 µg total RNA as a template in a 20-µl reaction with 1 x TaqMan RT buffer, 2.5 µM random hexamers, 5.5 mM magnesium chloride, 500 µM of each dNTP, 8 U RNase inhibitor, and 25 U MultiScribe reverse transcriptase. The reactions were incubated at 25C for 10 min and 48C for 30 min, followed by 95C for 5 min. The surrounding tissues of the ribs were scraped off and used for cDNA synthesis to detect possible DTDST expression in these structures and thus soft tissue contamination in cartilage cDNA PCR.
PCR assays were performed in 25-µl volumes using 1.0 µl cDNA as template, 1 µM of each primer, 1 x reaction buffer provided by the enzyme supplier, 0.28 mM of each nucleotide, and 0.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems). The following PCR conditions were used: 94C for 5 min, 37 cycles of 94C for 1 min, 57C for 1 min, and 72C for 1 min, followed by 72C for 8 min. The 542-bp product corresponds to nucleotides 5431084 of the published cDNA sequence (GenBank# U14528).
Northern Blot Hybridization
Commercially available Human and Human IV Multiple Tissue Northern Blots (Clontech) were hybridized with a 32P-labeled 542-bp (nucleotides 5431084) SLC26A2 cDNA fragment that was generated by PCR amplification using human lung cDNA as a template. After prehybridization overnight at 65C, the blots were hybridized for 18 hr at 65C in 10% dextran sulfate, 1 M NaCl, 1% SDS, and 160 µg/ml denatured salmon sperm DNA. After hybridization, the filters were washed at 65C in 3 x SSC, 0.1% SDS. Autoradiography was performed on X-ray films overnight at -20C. Equal loading was verified with control gene hybridization (data not shown).
Tissues
Formalin fixed, paraffin-embedded archival specimens from adult patients were obtained from the Department of Pathology, Haartman Institute, University of Helsinki (adult tissues) and Department of Pathology, University of Oulu (fetal tissues). ISH and IHC studies on fetal samples were approved by the ethics committees of the Departments of Medical Genetics and Dermatology. The following adult specimens were examined: endometrium at different menstrual phases (n=5), ventricle (n=4), duodenum (n=3), jejunum (n=2), ileum (n=1), appendix (n=2), colon (n=7), sigmoid (n=2), anal canal (n=2), liver (n=3), pancreas (n=4), skin (n=8), heart (n=3), bronchus (n=4), kidney (n=3), prostate (n=3), placenta (n=6), testis (n=4), thymus (n=4), epididymis (n=2), and articular cartilage (n=3). Fetal rib cartilage (n=2), complete fetuses at gestational ages of 67, 8, 1012 weeks and selected tissues from older fetuses at 11 and 20 weeks were also studied.
In Situ Hybridization
A 549-bp fragment corresponding to bases 14221970 of the published human SLC26A2 cDNA (GenBank#
U14528) was generated by PCR. This fragment was designed with a T7 RNA polymerase promoter at the 3' end and SP6 RNA polymerase promoter at the 5' end. Both sense and antisense probes were transcribed from the PCR product using the Riboprobe in vitro transcription system (Promega; Madison, WI). Antisense and sense RNA probes were labeled with -[35S]-UTP and purified probes were used at 4 x 105 cpm/liter of hybridization solution.
As previously described (
Normal colon samples known to be positive were used as controls in each experiment and a sense RNA probe was used as a negative control (
Immunohistochemistry
Antisera were raised in rabbits against two synthetic peptides ERQEKSDTNFKEFVIK and TVRDSLTNGEYCKKEEEN, corresponding to bases 196243 and 20922145, respectively, of the published cDNA sequence (GenBank#
U14528). Peptide synthesis and antibody production were purchased from Research Genetics (Huntsville, AL).
The specificity of antibodies was demonstrated by Western blotting (Fig 2) using homogenized osteosarcoma tissue. After centrifugation at 12,000 x g for 10 min, the supernatant was collected and diluted 1:4 in Laemmli sample buffer (Pharmacia; Uppsala, Sweden) containing 5% of ß-mercaptoethanol. Denatured proteins were separated on a 7.5% polyacrylamide gel and the gel was blotted onto Hybond C-extra (Amersham) membrane using standard protocols. Affinity-purified primary antibodies at 2 µg/ml were used. Normal rabbit IgG (Dako; Glostrup, Denmark) was used as a negative control antibody. Peroxidase-conjugated anti-rabbit IgG was diluted 1:10,000 in 0.1% Tween-20/PBS containing 2.5% non-fat milk and was used as the secondary antibody. The protein bands were visualized by chemiluminescence according to standard protocols.
|
|
Serial sections to those used for ISH were used for immunohistochemistry. The peroxidaseantiperoxidase technique was performed using the Vectastain Elite ABC Kit (Vector Laboratories; Burlingame, CA). For pretreatment, the deparaffinized slides were boiled in a microwave oven for 5 min in 10 mM citrate buffer (pH 6.0) or 0.01 M EDTA buffer (pH 8.0). One percent SDS for 5 min was used as a pretreatment for skin sections containing eccrine sweat glands. Anti-SLC26A2 sera were diluted 1:25001:4000. Diaminobenzidine (DAB) was used as the chromogenic substrate and the slides were counterstained with hematoxylin. Preimmune serum was used as a negative control on parallel sections.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Panel PCR
Intense bands were amplified from lung, placenta, and colon cDNAs and weaker bands from pancreas, kidney, and testis cDNAs of the multiple-tissue cDNA panel (MTC panel, Clontech; Fig 1). Faint bands could be detected in brain, heart, liver, peripheral blood leukocyte, small intestine, spleen, and thymus. Fetal cartilage cDNA was used as a positive control and demonstrated a single band with identical electrophoretic mobility (Fig 1).
Northern Hybridization
Northern analysis showed that the 542-bp SLC26A2 probe detects a single approximately 8.4-kb transcript, as expected (Fig 1). Strong signal was detected in placenta, prostate, testis, and colon, and weak signal in all the other tissues. The result corresponds to earlier Northern analysis (
Specificity of Anti-SLC26A2 Antiserum
Western blotting of osteosarcoma lysate using affinity-purified antibodies demonstrated a band with mobility corresponding approximately to the size of 90 kD (Fig 2). Anti-rabbit IgG used as a negative control did not detect this band. In addition, both SLC26A2-specific antibodies as well as normal anti-rabbit IgG recognized two smaller bands.
Immunohistochemistry
Cartilage.
Intense SLC26A2 mRNA expression was seen in hyaline cartilage of developing long bone of the limb in an 11-week-old fetus. The hypertrophic chondrocytes in the diaphysic maturation zones of limb long bones demonstrated strong SLC26A2 mRNA expression (Fig 3A and Fig 3B). The SLC26A2 protein expression was detected in the hypertrophic and proliferative chondrocytes in developing bones of an 11-week-old fetus (Fig 3C and Fig 3D). Fetal periosteum also demonstrated focal immunoreactivity for the SLC26A2 protein (Fig 3C). In addition, chondrocytes in adult bronchial cartilage showed mRNA expression (Fig 3F3I).
|
Colon.
We have previously shown that in normal colon SLC26A2 mRNA is detected in the upper one third of the crypt epithelium, mainly in the absorptive epithelial and goblet cells, whereas the signal was absent both at the bottom of the crypts and in luminal surface epithelium (
|
Placenta. Trophoblasts and syncytiotrophoblasts covering the surface of the chorionic villi demonstrated abundant SLC26A2 mRNA (Fig 5A5C) and protein expression (Fig 5D). Both strong cytoplasmic and apical plasma membrane immunoreactivity for the SLC26A2 protein could be detected, whereas the villous stromal tissue was negative (Fig 5D).
|
Sweat Gland. Eccrine sweat glands showed both SLC26A2 protein (Fig 6A) and mRNA (Fig 6D and Fig 6E) expression. Abundant immunoreactivity was detected in epithelial cells in the coiled secretory portion of the eccrine sweat gland (Fig 6C).
|
Pancreas and Liver. In the pancreas, acinar cells and duct epithelium of large ducts demonstrated strong immunoreactivity (Fig 7A, Fig 7B, Fig 7D, and Fig 7E). By contrast, cells of the islets of Langerhans were negative (Fig 7D). In addition, protein or cell debris in acinar lumen secretion stained with the SLC26A2 antiserum (Fig 7D). In the liver, signal for the SLC26A2 mRNA was detected only in occasional cells and no specific immunostaining for the protein could be seen (data not shown).
|
Bronchial Sections. High expression levels of both SLC26A2 mRNA and protein were detected in submucosal seromucous glands in the airway section of human bronchi (Fig 7F7H). SLC26A2 expression was also observed in the tracheal surface epithelium (Fig 7I and Fig 7J).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this work we present the expression patterns of human SLC26A2 (also known as DTDST) by ISH and IHC in vivo in different tissues and cell types. This is the first study demonstrating the expression at the cellular level in morphologically preserved tissues. SLC26A2 is responsible for four chondrodysplasias if mutated, and its function as a sulfate and chloride transporter has been established (
As expected, SLC26A2 expression was detected in several tissues. Cartilage, colon, placenta, bronchial glands, tracheal epithelium, pancreas, and eccrine sweat glands demonstrated immunoreactivity for the protein. Expression was observed in many different cell types but was confined mostly to secretory structures. Tissues such as testis, thymus, and prostate remained negative, and these results probably indicate that SLC26A2 expression level is so low that it escapes the ISH and IHC methods, although Northern analysis suggested expression. Expression can also be localized in a small, restricted area or structure. It is possible that the expression is variable and is regulated by certain events, whereas basal mRNA expression under normal physiological conditions is weak and the protein is not translated.
Interestingly, two antisera targeted against distinct parts of the SLC26A2 protein recognized expression in different tissues. Antiserum against the carboxy terminal peptide recognized expression in colon, placenta, bronchial glands, and tracheal epithelium, whereas antiserum against the amino terminal peptide recognized SLC26A2 expression in fetal cartilage, pancreas, and eccrine sweat glands. Both antisera demonstrated SLC26A2 protein expression in pancreatic ducts. However, under denaturing conditions in Western blotting analysis, both antisera recognized the same 90-kD mobility fragment in homogenized osteosarcoma tissue. In addition, no discrepancy between SLC26A2 mRNA expression by ISH and protein expression could be detected in this study.
Whereas SLC26A2 expression levels in adult cartilage were almost undetectable, strong signal for mRNA was observed in most mature hypertrophic chondrocytes at gestational Week 11 in developing fetal cartilage. In addition to hypertrophic chondrocytes, the protein immunostaining was detected in proliferative chondrocytes, whereas the primitive reserve chondrocytes were negative. The SLC26A2 protein expression in proliferative chondrocytes corresponds well with their active biosynthesis of sulfated proteoglycans and thus with the undersulfation of proteoglycans detected in patients with the defective SLC26A2 transporter. The sulfation of the chondroitin sulfate increases constantly with gestational age. At gestational Week 11, 6-sulfation, which is known to be more sensitive to extracellular inorganic sulfate depletion (
In addition to cartilage, SLC26A2 expression was detected in many different tissues and cell types that are not known to be affected in the four chondrodysplasias. In our earlier work, SLC26A2 mRNA was shown to be expressed in the upper one third of colonic crypt epithelium (
Other tissues that showed SLC26A2 mRNA and protein expression included placental villi, eccrine sweat glands, airway submucosal glands, and tracheal epithelial cells. In addition, protein expression was detected in exocrine pancreas. The presence of an active sulfate transport mechanism or even an SO42-/Cl- exchanger, as well as sulfated macromolecules in these tissues, has been reported (
The results reported here represent important steps towards defining the role of SLC26A2 (formerly DTDST) in anion transport processes of multiple tissues. SLC26A2 expression in cartilage and the phenotype caused by its defects establish its major role in providing enough inorganic sulfate for abundant biosynthesis of sulfated macromolecules in chondrocytes. However, it is still surprising that the phenotype of diastrophic dysplasia is restricted to cartilage and bone, although in vivo protein expression can be detected in many tissues. Many possible explanations exist. The residual activity of the mutated SLC26A2 protein combined with the use of alternative sulfate sources (
![]() |
Acknowledgments |
---|
Supported by the Helsinki University Research Fund, the Academy of Finland, The Finnish Medical Foundation, the Duodecim Foundation, the Research and Science Foundation of Farmos, the Sigrid Juselius Foundation, and the Finnish Pediatric Foundation, Ulla Hjelt Fund.
We thank Dr Riitta Herva and Dr Juha-Pekka Turunen for their pathology expertise. The skillful technical assistance of Ms Alli Tallqvist and Ms Ranja Eklund is gratefully acknowledged.
Received for publication September 28, 2000; accepted February 20, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cole DE, Rastogi N (1991) Sulfate transport in human placenta: further evidence for a sodium-independent mechanism. Biochim Biophys Acta 1064:287-292[Medline]
Elgavish A, DiBona DR, Norton P, Meezan E (1987) Sulfate transport in apical membrane vesicles isolated from tracheal epithelium. Am J Physiol 253:C416-425
Elgavish A, Meezan E (1992) Altered sulfate transport via anion exchange in CFPAC is corrected by retrovirus-mediated CFTR gene transfer. Am J Physiol 263:C176-186
Esko JD, Elgavish A, Prasthofer T, Taylor WH, Weinke JL (1986) Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J Biol Chem 261:15725-15733
Everett LA, Glaser B, Beck JC, Idol JR, Buchs A, Heyman M, Adawi F, Hazani E, Nassir E, Baxevanis AD, Sheffield VC, Green ED (1997) Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nature Genet 17:411-422[Medline]
Everett LA, Morsli H, Wu DK, Green ED (1999) Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear. Proc Natl Acad Sci USA 96:9727-9732
Haila S, SaarialhoKere U, KarjalainenLindsberg M-L, Lohi H, Airola K, Holmberg C, Hästbacka J, Kere J, Hoglund P (2000) The congenital chloride diarrhea gene is expressed in seminal vesicle, sweat gland, inflammatory colon epithelium, and in some dysplastic colon cells. Histochem Cell Biol 113:279-286[Medline]
Hästbacka J, de la Chapelle A, Mahtani MM, Clines G, ReeveDaly MP, Daly M, Hamilton BA, Kusumi K, Trivedi B, Weaver A, Coloma A, Lovett M, Buckler A, Kaitila I, Lander ES (1994) The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping. Cell 78:1073-1087[Medline]
Hästbacka J, SupertiFurga A, Wilcox WR, Rimoin DL, Cohn DH, Lander ES (1996) Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for phenotypic series involving three chondrodysplasias. Am J Hum Genet 58:255-262[Medline]
Höglund P, Haila S, Socha J, Tomaszewski L, SaarialhoKere U, KarjalainenLindsberg M-L, Airola K, Holmberg C, de la Chapelle A, Kere J (1996) Mutations of the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea. Nature Genet 14:316-319[Medline]
Ito K, Kimata K, Sobue M, Suzuki S (1982) Altered proteoglycan synthesis by epiphyseal cartilages in culture at low SO42- concentration. J Biol Chem 257:917-923
Lohi H, Kujala M, Kerkelä E, SaarialhoKere U, Kestilä M, Kere J (2000) Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger. Genomics 70:102-112[Medline]
Parmley RT, Takagi M, Denys FR (1984) Ultrastructural localization of glycosaminoglycans in human term placenta. Anat Rec 210:477-484[Medline]
Prosser IW, Stenmark KR, Suthar M, Crouch EC, Mecham RP, Parks WC (1989) Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am J Pathol 135:1073-1088[Abstract]
Pye DA, Vives RR, Turnbull JE, Hyde P, Gallagher JT (1998) Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J Biol Chem 273:22936-22942
Rossi A, Bonaventure J, Delezoide AL, Cetta G, SupertiFurga A (1996) Undersulfation of proteoglycans synthesized by chondrocytes from a patient with achondrogenesis type 1B homozygous for an L483P substitution in the diastrophic dysplasia sulfate transporter. J Biol Chem 271:18456-18464
Roughley PJ, White RJ, Glant TT (1987) The structure and abundance of cartilage proteoglycans during early development of the human fetus. Pediatr Res 22:409-413[Abstract]
Satoh H, Susaki M, Shukunami C, Iyama K, Negoro T, Hiraki Y (1998) Functional analysis of diastrophic dysplasia sulfate transporter. Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans. J Biol Chem 273:12307-12315
Scott DA, Karniski LP (2000) Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am J Physiol Cell Physiol 278:C207-211
Scott DA, Wang R, Kreman TM, Sheffield VC, Karnishki LP (1999) The Pendred syndrome gene encodes a chloride-iodide transport protein. Nature Genet 21:440-443[Medline]
Seutter E, Trijbels JM, Sutorius AH, Urselmann EJ (1970) The sweat gland as a mucous gland. Dermatologica 141:397-408[Medline]
SupertiFurga A, Hästbacka J, Wilcox WR, Cohn DH, van der Harten HJ, Rossi A, Blau N, Rimoin DL, Steinmann B, Lander ES, Gitzelmann R (1996) Achondrogenesis type IB is caused by mutations in the diastrophic dysplasia sulphate transporter gene. Nature Genet 12:100-102[Medline]
SupertiFurga A, Neumann L, Riebel T, Eich G, Steinmann B, Spranger J, Kunze J (1999) Recessively inherited multiple epiphyseal dysplasia with normal stature, club foot, and double layered patella caused by a DTDST mutation. J Med Genet 36:621-624
Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P (2000) Prestin is the motor protein of cochlear outer hair cells. Nature 405:149-155[Medline]