CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression... more CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon. Involvement of CRF(1) receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G(i)-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.
Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study ge... more Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vectormediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.
frameshift with premature translational termination at The reeler Albany2 mutation (Reln rl-Alb2 ... more frameshift with premature translational termination at The reeler Albany2 mutation (Reln rl-Alb2 ) in the mouse nucleotide 5967 of the normal, 11,673-nucleotide-long is an allele of reeler isolated during a chlorambucil sequence and in a drastic reduction of reelin mRNA conmutagenesis screen. Homozygous animals had drasticentration. To our knowledge, this is the first example cally reduced concentrations of reelin mRNA, in which of an insertion of an IAP element following chemical an 85-nt exon was absent. At the genomic level, the mutagenesis. mutation was shown to be due to an intracisternal Aparticle insertion leading to exon skipping. This appears to be the first observation of retrotransposon METHODS insertion during chlorambucil mutagenesis. ᭧ 1997 Aca-
generated in normal numbers and at the normal time, Reelin is the protein defective in reeler mic... more generated in normal numbers and at the normal time, Reelin is the protein defective in reeler mice, an exand their radial migration begins normally. However, tensively studied model of brain development. The at the end of migration, neurons fail to form normal reelin gene (symbol Reln) codes for a protein of the patterns. The abnormality is most evident in the cereextracellular matrix that contains eight successive rebellum, where it leads to gross atrophy with loss of peats of 350 to 390 amino acids. In this work, we degranule cells, and it affects several structures of the scribe the genomic structure of the mouse reelin gene central nervous system, particularly the cerebral corand the 5-flanking genomic DNA sequences. The reelin tex and hippocampus (Caviness and Rakic, 1978; Gofgene is composed of 65 exons spread over approxifinet, 1984).
Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain developme... more Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain development. However, some uncertainties remain about the relationship between reelin and the reeler phenotype. It is generally believed that reelin, secreted by specific neuronal types such as Cajal-Retzius cells, acts at short distance via the extracellular matrix on target neurons, the response of which requires the Dab1 gene product. However, the pattern of reelin expression in some structures such as olfactory bulb, retina, and spinal cord suggests that the protein might be endowed with different functions. In the present study, we identify two uncommon, evolutionarily conserved splicing events in the 3Ј part of the transcript that result in different forms of the protein. First, a 6-nucleotide, brain-specific microexon is skipped in about 10% of reelin RNA. In addition, an alternative polyadenylation event involving 10-25% of reelin mRNA results in secretion of a truncated protein lacking the terminal, highly basic stretch. This alternative reelin is generally expressed in the same cells as the major form, but is almost undetectable in retina and spinal cord. Both alternative splicing events are present in mouse, rat, and man, suggesting that the corresponding reelin forms are functionally important. 1999 Academic Press
The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in... more The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development and evolution. No gene product is known, and the locus is currently being characterized by positional cloning. YAC clones corresponding to the closest markers &I&fit61 and D5iWt72 have been isolated. Cloned extremities of the YAC inserts were used to construct a 1.1.Mb contig, a 700.kb fragment of which was shown to contain the reeler locus. The integrity of the contig was verified by physical mapping on genomic DNA. The classical allele of the reeler mutation was associated with a 150.kb deletion between D52Mit61 and D5Mit72, while no gross chromosomal anomaly was found in the Orleans allele. Candidate coding sequences were isolated to construct a preliminary transcriptional map of the reeler region. Cosmid clones mapping within the rl deletion revealed a large transcript of more than 11 kb, which was present in normal embryonic brain but barely detectable in homoxygous rP1lrZorl embryonic brain, suggesting strongly that it corresponds to the reeler transcript.
The lumen of the thyroid follicle is necessary for the maximal accumulation and organification of... more The lumen of the thyroid follicle is necessary for the maximal accumulation and organification of iodide, as well as the synthesis of thyroid hormones that are required for the homeostasis of all cells in terrestrial mammals. Polarized thyrocytes surround the follicular lumen; with their basal and apical surfaces facing the bloodstream and the lumen, respectively 1 . Iodide is concentrated at the basolateral membrane by the sodium iodide symporter (NIS) 2 and appears to be ported into the follicular lumen by pendrin, the product of the Pendred syndrome gene (PDS ), which is located on the apical membrane 3 . Once synthesized, thyroglobulin (TG), the major product of the thyrocyte, is vectorially transported to the apical membrane and released into the follicular lumen, where it becomes the macromolecular storage site for iodine 1,4 . Thyroid peroxidase (TPO) and hydrogen peroxide, located at the apical membrane, catalyze the iodination of Tyr residues on TG, and then couple these iodotyrosine residues to form thyroxine (T 4 ) and triiodothyroxine (T 3 ) 1,4,5 . Iodinated TG is resorbed through the apical membrane and degraded to form T 3 /T 4 in lysosomes; the T 3 /T 4 is then secreted through the basal membrane 1,4,5 .
Pendred syndrome is an autosomal recessive disorder characterized by congenital deafness and thyr... more Pendred syndrome is an autosomal recessive disorder characterized by congenital deafness and thyroid goiter. The thyroid disease typically develops around puberty and is associated with a mild organification defect, characterized by an inappropriate discharge of iodide upon perchlorate stimulation (a positive perchlorate discharge test). The gene (PDS) mutated in Pendred syndrome is expressed in thyroid and encodes a 780-amino acid protein (pendrin) that has recently been shown to function as an iodide/chloride transporter. We sought to establish the location of pendrin in the thyroid and to examine the regulatory network controlling its synthesis. Using peptide-specific antibodies for immunolocalization studies, pendrin was detected in a limited subset of cells within the thyroid follicles, ex-clusively at the apical membrane of the follicular epithelium. Interestingly, significantly greater amounts of pendrin were encountered in thyroid tissue from patients with Graves' disease. Using a cultured rat thyroid cell line (FRTL-5), PDS expression was found to be significantly induced by low concentrations of thyroglobulin (TG), but not by TSH, sodium iodide, or insulin. This is different from the established effect of TG, more typically a potent suppressor of thyroid-specific gene expression. Together, these results suggest that pendrin is an apical porter of iodide in the thyroid and that the expression and function of both the apical and basal iodide porters are coordinately regulated by follicular TG. (Endocrinology 141: 839 -845, 2000)
Proceedings of The National Academy of Sciences, 2001
Pendrin is an anion transporter encoded by the PDS͞Pds gene. In humans, mutations in PDS cause th... more Pendrin is an anion transporter encoded by the PDS͞Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS͞Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H ؉ -ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H ؉ -ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.
Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appea... more Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl Ϫ /HCO 3 Ϫ exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na ϩ -K ϩ -ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl Ϫ /HCO 3 Ϫ exchange in gills of Atlantic stingrays from both freshwater and marine environments.
Pendred syndrome is a major cause of congenital deafness, goiter and defective iodide organificat... more Pendred syndrome is a major cause of congenital deafness, goiter and defective iodide organification. Mutations in the transmembrane protein, pendrin, cause diminished export of iodide from thyroid follicular cells to the colloid and are associated with the syndrome. We used green fluorescent protein (GFP) chimeras of wild-type (WT) pendrin and three common natural mutants (L236P, T416P and G384) to study their intracellular trafficking in living cells. Time-lapse imaging, dual color labeling and fluorescent recovery after photobleaching (FRAP) studies demonstrated that GFP-WT pendrin targets to the plasma membrane. In contrast, all three mutant pendrins were retained in the endoplasmic reticulum (ER) in co-localization studies with ER and Golgi markers. The ER retention of L236P appeared to be selective as this mutant did not prevent a viral membrane protein, VSVGtsO45 or wild-type pendrin from targeting the plasma membrane. These findings suggest that ER retention and defective plasma membrane targeting of pendrin mutants play a key role in the pathogenesis of Pendred syndrome.
Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic v... more Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO 3 and Cl -, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (ϩ/ϩ) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (ϩ/ϩ) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (ϩ/ϩ) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis. (Hypertension. 2003;42: 356-362.)
Background: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital d... more Background: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4 +/+ ) or lack a complete Slc26a4 gene (Slc26a4 -/-).
Jaro-journal of The Association for Research in Otolaryngology, 2003
Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the ... more Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pdsknockout (Pds )/) ) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds )/) mice was only two-thirds the thickness seen in wildtype mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds )/) mice. Consistent with the altered strial morphology, the endocochlear potential in Pds )/) mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds )/) mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/ or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.
CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression... more CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon. Involvement of CRF(1) receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G(i)-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.
Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study ge... more Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vectormediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.
frameshift with premature translational termination at The reeler Albany2 mutation (Reln rl-Alb2 ... more frameshift with premature translational termination at The reeler Albany2 mutation (Reln rl-Alb2 ) in the mouse nucleotide 5967 of the normal, 11,673-nucleotide-long is an allele of reeler isolated during a chlorambucil sequence and in a drastic reduction of reelin mRNA conmutagenesis screen. Homozygous animals had drasticentration. To our knowledge, this is the first example cally reduced concentrations of reelin mRNA, in which of an insertion of an IAP element following chemical an 85-nt exon was absent. At the genomic level, the mutagenesis. mutation was shown to be due to an intracisternal Aparticle insertion leading to exon skipping. This appears to be the first observation of retrotransposon METHODS insertion during chlorambucil mutagenesis. ᭧ 1997 Aca-
generated in normal numbers and at the normal time, Reelin is the protein defective in reeler mic... more generated in normal numbers and at the normal time, Reelin is the protein defective in reeler mice, an exand their radial migration begins normally. However, tensively studied model of brain development. The at the end of migration, neurons fail to form normal reelin gene (symbol Reln) codes for a protein of the patterns. The abnormality is most evident in the cereextracellular matrix that contains eight successive rebellum, where it leads to gross atrophy with loss of peats of 350 to 390 amino acids. In this work, we degranule cells, and it affects several structures of the scribe the genomic structure of the mouse reelin gene central nervous system, particularly the cerebral corand the 5-flanking genomic DNA sequences. The reelin tex and hippocampus (Caviness and Rakic, 1978; Gofgene is composed of 65 exons spread over approxifinet, 1984).
Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain developme... more Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain development. However, some uncertainties remain about the relationship between reelin and the reeler phenotype. It is generally believed that reelin, secreted by specific neuronal types such as Cajal-Retzius cells, acts at short distance via the extracellular matrix on target neurons, the response of which requires the Dab1 gene product. However, the pattern of reelin expression in some structures such as olfactory bulb, retina, and spinal cord suggests that the protein might be endowed with different functions. In the present study, we identify two uncommon, evolutionarily conserved splicing events in the 3Ј part of the transcript that result in different forms of the protein. First, a 6-nucleotide, brain-specific microexon is skipped in about 10% of reelin RNA. In addition, an alternative polyadenylation event involving 10-25% of reelin mRNA results in secretion of a truncated protein lacking the terminal, highly basic stretch. This alternative reelin is generally expressed in the same cells as the major form, but is almost undetectable in retina and spinal cord. Both alternative splicing events are present in mouse, rat, and man, suggesting that the corresponding reelin forms are functionally important. 1999 Academic Press
The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in... more The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development and evolution. No gene product is known, and the locus is currently being characterized by positional cloning. YAC clones corresponding to the closest markers &I&fit61 and D5iWt72 have been isolated. Cloned extremities of the YAC inserts were used to construct a 1.1.Mb contig, a 700.kb fragment of which was shown to contain the reeler locus. The integrity of the contig was verified by physical mapping on genomic DNA. The classical allele of the reeler mutation was associated with a 150.kb deletion between D52Mit61 and D5Mit72, while no gross chromosomal anomaly was found in the Orleans allele. Candidate coding sequences were isolated to construct a preliminary transcriptional map of the reeler region. Cosmid clones mapping within the rl deletion revealed a large transcript of more than 11 kb, which was present in normal embryonic brain but barely detectable in homoxygous rP1lrZorl embryonic brain, suggesting strongly that it corresponds to the reeler transcript.
The lumen of the thyroid follicle is necessary for the maximal accumulation and organification of... more The lumen of the thyroid follicle is necessary for the maximal accumulation and organification of iodide, as well as the synthesis of thyroid hormones that are required for the homeostasis of all cells in terrestrial mammals. Polarized thyrocytes surround the follicular lumen; with their basal and apical surfaces facing the bloodstream and the lumen, respectively 1 . Iodide is concentrated at the basolateral membrane by the sodium iodide symporter (NIS) 2 and appears to be ported into the follicular lumen by pendrin, the product of the Pendred syndrome gene (PDS ), which is located on the apical membrane 3 . Once synthesized, thyroglobulin (TG), the major product of the thyrocyte, is vectorially transported to the apical membrane and released into the follicular lumen, where it becomes the macromolecular storage site for iodine 1,4 . Thyroid peroxidase (TPO) and hydrogen peroxide, located at the apical membrane, catalyze the iodination of Tyr residues on TG, and then couple these iodotyrosine residues to form thyroxine (T 4 ) and triiodothyroxine (T 3 ) 1,4,5 . Iodinated TG is resorbed through the apical membrane and degraded to form T 3 /T 4 in lysosomes; the T 3 /T 4 is then secreted through the basal membrane 1,4,5 .
Pendred syndrome is an autosomal recessive disorder characterized by congenital deafness and thyr... more Pendred syndrome is an autosomal recessive disorder characterized by congenital deafness and thyroid goiter. The thyroid disease typically develops around puberty and is associated with a mild organification defect, characterized by an inappropriate discharge of iodide upon perchlorate stimulation (a positive perchlorate discharge test). The gene (PDS) mutated in Pendred syndrome is expressed in thyroid and encodes a 780-amino acid protein (pendrin) that has recently been shown to function as an iodide/chloride transporter. We sought to establish the location of pendrin in the thyroid and to examine the regulatory network controlling its synthesis. Using peptide-specific antibodies for immunolocalization studies, pendrin was detected in a limited subset of cells within the thyroid follicles, ex-clusively at the apical membrane of the follicular epithelium. Interestingly, significantly greater amounts of pendrin were encountered in thyroid tissue from patients with Graves' disease. Using a cultured rat thyroid cell line (FRTL-5), PDS expression was found to be significantly induced by low concentrations of thyroglobulin (TG), but not by TSH, sodium iodide, or insulin. This is different from the established effect of TG, more typically a potent suppressor of thyroid-specific gene expression. Together, these results suggest that pendrin is an apical porter of iodide in the thyroid and that the expression and function of both the apical and basal iodide porters are coordinately regulated by follicular TG. (Endocrinology 141: 839 -845, 2000)
Proceedings of The National Academy of Sciences, 2001
Pendrin is an anion transporter encoded by the PDS͞Pds gene. In humans, mutations in PDS cause th... more Pendrin is an anion transporter encoded by the PDS͞Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS͞Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H ؉ -ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H ؉ -ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.
Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appea... more Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl Ϫ /HCO 3 Ϫ exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na ϩ -K ϩ -ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl Ϫ /HCO 3 Ϫ exchange in gills of Atlantic stingrays from both freshwater and marine environments.
Pendred syndrome is a major cause of congenital deafness, goiter and defective iodide organificat... more Pendred syndrome is a major cause of congenital deafness, goiter and defective iodide organification. Mutations in the transmembrane protein, pendrin, cause diminished export of iodide from thyroid follicular cells to the colloid and are associated with the syndrome. We used green fluorescent protein (GFP) chimeras of wild-type (WT) pendrin and three common natural mutants (L236P, T416P and G384) to study their intracellular trafficking in living cells. Time-lapse imaging, dual color labeling and fluorescent recovery after photobleaching (FRAP) studies demonstrated that GFP-WT pendrin targets to the plasma membrane. In contrast, all three mutant pendrins were retained in the endoplasmic reticulum (ER) in co-localization studies with ER and Golgi markers. The ER retention of L236P appeared to be selective as this mutant did not prevent a viral membrane protein, VSVGtsO45 or wild-type pendrin from targeting the plasma membrane. These findings suggest that ER retention and defective plasma membrane targeting of pendrin mutants play a key role in the pathogenesis of Pendred syndrome.
Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic v... more Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO 3 and Cl -, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (ϩ/ϩ) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (ϩ/ϩ) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (ϩ/ϩ) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis. (Hypertension. 2003;42: 356-362.)
Background: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital d... more Background: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4 +/+ ) or lack a complete Slc26a4 gene (Slc26a4 -/-).
Jaro-journal of The Association for Research in Otolaryngology, 2003
Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the ... more Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pdsknockout (Pds )/) ) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds )/) mice was only two-thirds the thickness seen in wildtype mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds )/) mice. Consistent with the altered strial morphology, the endocochlear potential in Pds )/) mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds )/) mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/ or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.
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Papers by Ines Royaux