Philine Wangemann

Kansas State University, Kansas, United States

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Publications (102)345.83 Total impact

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    ABSTRACT: In the mammalian inner ear, bicellular and tricellular tight junctions seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tricellular tight junctions (tTJs) of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC encoding tricellulin (DFNB49) and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells but have a normal endocochlear potential (EP). ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in ILDR1 null mice after the first postnatal week. As revealed by freeze fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory hair cells.
    Human Molecular Genetics 09/2014; · 7.69 Impact Factor
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    ABSTRACT: SLC26A4 mutations can cause a distinctive hearing loss phenotype with sudden drops and fluctuation in patients. Existing Slc26a4 mutant mouse lines have a profound loss of hearing and vestibular function, with severe inner ear malformations that do not model this human phenotype. In this study, we generated Slc26a4-insufficient mice by manipulation of doxycycline administration to a transgenic mouse line in which all Slc26a4 expression was under the control of doxycycline. Doxycycline was administered from conception to embryonic day 17.5, and then it was discontinued. Auditory brainstem response thresholds showed significant fluctuation of hearing loss from 1 through 3months of age. The endocochlear potential, which is required for inner ear sensory cell function, correlated with auditory brainstem response thresholds. We observed degeneration of stria vascularis intermediate cells, the cells that generate the endocochlear potential, but no other abnormalities within the cochlea. We conclude that fluctuations of hearing result from fluctuations of the endocochlear potential and stria vascularis dysfunction in Slc26a4-insufficient mouse ears. This model can now be used to test potential interventions to reduce or prevent sudden hearing loss or fluctuation in human patients. Our strategy to generate a hypomorphic mouse model utilizing the tet-on system will be applicable to other diseases in which a hypomorphic allele is needed to model the human phenotype.
    Neurobiology of Disease 02/2014; · 5.62 Impact Factor
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    ABSTRACT: Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner's membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner's membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO3- over Cl- and inhibited by I- and NPPB. Elevated NO3- currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl- transport in Reissner's membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl- conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner's membrane cells during local perturbations of pH.
    PLoS ONE 01/2014; 9(5):e97191. · 3.53 Impact Factor
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    ABSTRACT: Pendred syndrome (PS) is characterized by autosomal recessive inheritance of goiter associated with a defect of iodide organification, hearing loss, enlargement of the vestibular aqueduct (EVA), and mutations of the
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    ABSTRACT: Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (Δ/Δ) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (Δ/Δ) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
    PLoS Genetics 07/2013; 9(7):e1003641. · 8.52 Impact Factor
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    Xiangming Li, Fei Zhou, Daniel C Marcus, Philine Wangemann
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    ABSTRACT: Slc26a4 (Δ/Δ) mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na(+) and K(+) were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4 (Δ/+), which develop normal hearing, and of Slc26a4 (Δ/Δ) mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na(+) (∼141 mM) and low K(+) concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. Shortly before birth the K(+) concentration began to rise. Immediately after birth (postnatal day 0), the Na(+) and K(+) concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4 (Δ/Δ) mice, the rise in the K(+) concentration occurred with a ∼3 day delay. K(+) concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K(+) channel KCNQ1 and the Na(+)/2Cl(-)/K(+) cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na(+)-rich fluid, which transitions into a K(+)-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na(+) transport.
    PLoS ONE 01/2013; 8(5):e65977. · 3.53 Impact Factor
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    ABSTRACT: Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-N(G)-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca(2+) concentration and/or the Ca(2+) sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca(2+), and Ca(2+) sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K(+) and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca(2+) concentration in smooth muscle cells but increased the Ca(2+) sensitivity. This increase in the Ca(2+) sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca(2+) sensitivity and abolished the LNNA-induced increase in Ca(2+) sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca(2+) sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca(2+) sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.
    PLoS ONE 01/2013; 8(1):e53655. · 3.53 Impact Factor
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    ABSTRACT: Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.
    European Journal Of Oral Sciences 12/2011; 119 Suppl 1:185-92. · 1.42 Impact Factor
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    ABSTRACT: The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α(1A)-adrenergic receptor subtype mediates vasoconstriction of the gerbil spiral modiolar artery. Although the gerbil cochlea is often used as a model in hearing research, the molecular and pharmacological characteristics of the cloned gerbil α(1a)-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α(1a)-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α(1a)-adrenergic receptors. The cDNA clone contained 1404 nucleotides, which encoded a 467 amino acid peptide with a deduced sequence having 96.8, 96.4 and 91.6% identity to rat, mouse and human α(1a)-receptors, respectively. We transiently transfected the α(1a)-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [(3)H]prazosin binding. Unlabeled prazosin had a K(i) of 0.89±0.1nM. The α(1A)-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had K(i) values of 4.9±1 and 1.0±0.1nM, respectively. BMY-7378, an α(1D)-adrenergic receptor-selective antagonist, bound with low affinity (260±60nM). The 91.6% amino acid sequence identity and K(i)s of the cloned gerbil α(1a)-adrenergic receptor are similar to those of the human α(1a)-adrenergic receptor clone. These results show that the gerbil α(1a)-adrenergic receptor is representative of the human α(1a)-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.
    Hearing research 11/2011; 283(1-2):144-50. · 2.18 Impact Factor
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    ABSTRACT: Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3- into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.
    The Journal of clinical investigation 10/2011; 121(11):4516-25. · 15.39 Impact Factor
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    ABSTRACT: Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery. Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times. This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.
    BMC Physiology 08/2011; 11:15.
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    Andrew J Griffith, Philine Wangemann
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    ABSTRACT: Enlargement of the vestibular aqueduct (EVA) is one of the most common inner ear malformations associated with sensorineural hearing loss in children. The delayed onset and progressive nature of this phenotype offer a window of opportunity to prevent or retard progression of hearing loss. EVA is not the direct cause of hearing loss in these patients, but rather is a radiologic marker for some underlying pathogenetic defect. Mutations of the SLC26A4 gene are a common cause of EVA. Studies of an Slc26a4 knockout mouse demonstrate that acidification and enlargement of the scala media are early events in the pathogenesis of deafness. The enlargement is driven by fluid secretion in the vestibular labyrinth and a failure of fluid absorption in the embryonic endolymphatic sac. Elucidating the mechanism of hearing loss may offer clues to potential therapeutic strategies.
    Hearing research 06/2011; 281(1-2):11-7. · 2.18 Impact Factor
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    ABSTRACT: Enlargement of the vestibular aqueduct (EVA) is the most common inner ear anomaly detected in ears of children with sensorineural hearing loss. Pendred syndrome (PS) is an autosomal recessive disorder characterized by bilateral sensorineural hearing loss with EVA and an iodine organification defect that can lead to thyroid goiter. Pendred syndrome is caused by mutations of the SLC26A4 gene. SLC26A4 mutations may also be identified in some patients with nonsyndromic EVA (NSEVA). The presence of two mutant alleles of SLC26A4 is correlated with bilateral EVA and Pendred syndrome, whereas unilateral EVA and NSEVA are correlated with one (M1) or zero (M0) mutant alleles of SLC26A4. Thyroid gland enlargement (goiter) appears to be primarily dependent on the presence of two mutant alleles of SLC26A4 in pediatric patients, but not in older patients. In M1 families, EVA may be associated with a second, undetected SLC26A4 mutation or epigenetic modifications. In M0 families, there is probably etiologic heterogeneity that includes causes other than, or in addition to, monogenic inheritance.
    Cellular Physiology and Biochemistry 01/2011; 28(3):545-52. · 3.42 Impact Factor
  • Philine Wangemann
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    ABSTRACT: Enlargement of the vestibular aqueduct (EVA) is a common inner ear malformation found in children with sensorineural hearing loss that is frequently associated with loss-of-function or hypo-function mutations of SLC26A4. SLC26A4 codes for pendrin, which is a protein that is expressed in apical membranes of selected epithelia and functions as an anion exchanger. The comparatively high prevalence of EVA provides a strong imperative to develop rational interventions that delay, ameliorate or prevent hearing loss associated with this phenotype. The development of rational interventions requires a fundamental understanding of the role that pendrin plays in the normal development of hearing, as well as a detailed understanding of the pathobiologic mechanisms that, in the absence of fully functional pendrin, lead to an unstable hearing phenotype, with fluctuating or progressive loss of hearing. This review summarizes studies in mouse models that have focused on delineating the role of pendrin in the physiology of the inner ear and the pathobiology that leads to hearing loss.
    Cellular Physiology and Biochemistry 01/2011; 28(3):527-34. · 3.42 Impact Factor
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    Hyoung-Mi Kim, Philine Wangemann
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    ABSTRACT: Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.
    PLoS ONE 01/2011; 6(3):e17949. · 3.53 Impact Factor
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    ABSTRACT: Regulation of cochlear blood flow is critical for hearing due to its exquisite sensitivity to ischemia and oxidative stress. Many forms of hearing loss such as sensorineural hearing loss and presbyacusis may involve or be aggravated by blood flow disorders. Animal experiments and clinical outcomes further suggest that there is a gender preference in hearing loss, with males being more susceptible. Autoregulation of cochlear blood flow has been demonstrated in some animal models in vivo, suggesting that similar to the brain, blood vessels supplying the cochlea have the ability to control flow within normal limits, despite variations in systemic blood pressure. Here, we investigated myogenic regulation in the cochlear blood supply of the Mongolian gerbil, a widely used animal model in hearing research. The cochlear blood supply originates at the basilar artery, followed by the anterior inferior cerebellar artery, and inside the inner ear, by the spiral modiolar artery and the radiating arterioles that supply the capillary beds of the spiral ligament and stria vascularis. Arteries from male and female gerbils were isolated and pressurized using a concentric pipette system. Diameter changes in response to increasing luminal pressures were recorded by laser scanning microscopy. Our results show that cochlear vessels from male and female gerbils exhibit myogenic regulation but with important differences. Whereas in male gerbils, both spiral modiolar arteries and radiating arterioles exhibited pressure-dependent tone, in females, only radiating arterioles had this property. Male spiral modiolar arteries responded more to L-NNA than female spiral modiolar arteries, suggesting that NO-dependent mechanisms play a bigger role in the myogenic regulation of male than female gerbil cochlear vessels.
    PLoS ONE 01/2011; 6(9):e25659. · 3.53 Impact Factor
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    ABSTRACT: Bronckers ALJJ, Guo J, Zandieh-Doulabi B, Bervoets TJ, Lyaruu DM, Li X, Wangemann P, DenBesten P. Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth. Eur J Oral Sci 2011; 119 (Suppl. 1): 185-192. © 2011 Eur J Oral Sci Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.
    European Journal Of Oral Sciences 01/2011; 119(Suppl 1-119):185-92. · 1.42 Impact Factor
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    ABSTRACT: We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss. We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks. TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 μmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1(G82D)) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1(S225A)) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56 ± 0.20 weeks), which matched the kinetics predicted for a vascular origin. TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.
    Stroke 10/2010; 41(11):2618-24. · 6.16 Impact Factor
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    ABSTRACT: Mass spectrometry (MS) has become the method of choice for protein/peptide sequence and modification analysis. The technology employs a two-step approach: ionized peptide precursor masses are detected, selected for fragmentation, and the fragment mass spectra are collected for computational analysis. Current precursor selection schemes are based on data- or information-dependent acquisition (DDA/IDA), where fragmentation mass candidates are selected by intensity and are subsequently included in a dynamic exclusion list to avoid constant refragmentation of highly abundant species. DDA/IDA methods do not exploit valuable information that is contained in the fractional mass of high-accuracy precursor mass measurements delivered by current instrumentation. We extend previous contributions that suggest that fractional mass information allows targeted fragmentation of analytes of interest. We introduce a non-linear Random Forest classification and a discrete mapping approach, which can be trained to discriminate among arbitrary fractional mass patterns for an arbitrary number of classes of analytes. These methods can be used to increase fragmentation efficiency for specific subsets of analytes or to select suitable fragmentation technologies on-the-fly. We show that theoretical generalization error estimates transfer into practical application, and that their quality depends on the accuracy of prior distribution estimate of the analyte classes. The methods are applied to two real-world proteomics datasets. All software used in this study is available from http://software.steenlab.org/fmf hanno.steen@childrens.harvard.edu Supplementary data are available at Bioinformatics online.
    Bioinformatics 03/2010; 26(6):791-7. · 5.47 Impact Factor
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    Hyoung-Mi Kim, Philine Wangemann
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    ABSTRACT: Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4(-/-), results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4(+/-) and Slc26a4(-/-) mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4(-/-) compared to Slc26a4(+/-) mice. In Slc26a4(+/-) and Slc26a4(-/-) mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4(+/-) mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4(-/-) mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.
    PLoS ONE 01/2010; 5(11):e14041. · 3.53 Impact Factor

Publication Stats

3k Citations
345.83 Total Impact Points

Institutions

  • 1999–2014
    • Kansas State University
      • Department of Anatomy and Physiology
      Kansas, United States
  • 2007–2013
    • University of Tuebingen
      Tübingen, Baden-Württemberg, Germany
  • 2011
    • National Institutes of Health
      • Molecular Targets Laboratory
      Bethesda, MD, United States
  • 2003
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1989–1998
    • Boys Town National Research Hospital
      Omaha, Nebraska, United States
  • 1996
    • Boys Town National Research Hospital
      Omaha, Nebraska, United States
  • 1987
    • Max Planck Institute of Biophysics
      Frankfurt, Hesse, Germany