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ABSTRACT: The autosomal recessive mutation, sld, attenuates mucous cell expression in murine sublingual glands with corresponding effects on mucin 19 (Muc19). We conducted a systematic study including genetic mapping, sequencing and functional analyses to elucidate a mutation to explain the sld phenotype in neonatal mice. Genetic mapping and gene expression analyses localized the sld mutation within the gene Muc19/Smgc, specifically attenuating Muc19 transcripts, and Muc19 knockout mice mimic the sld phenotype in neonates. Muc19 transcription is unaffected in sld mice whereas mRNA stability is markedly decreased. Decreased mRNA stability is not due to a defect in 3 prime-end processing nor to sequence differences in Muc19 transcripts. Comparative sequencing of the Muc19/Smgc gene identified four candidate intronic mutations within the Muc19 coding region. Minigene splicing assays revealed a novel splicing event in which insertion of two additional repeats within a CA-repeat region of intron 53 of the sld genome enhances retention of intron 54, decreasing the levels of correctly spliced transcripts. Moreover, Pateamine A, an inhibitor of nonsense-mediated mRNA decay, inhibits degradation of aberrant Muc19 transcripts. The mutation in intron 53 thus enhances aberrant splicing leading to degradation of aberrant transcripts and decreased Muc19 message stability, consistent with the sld phenotype. We propose a working model of the unique splicing event enhanced by the mutation, as well as putative explanations for the gradual but limited increase in Muc19 glycoprotein expression and its restricted localization to subpopulations of mucous cells in sld mice during postnatal gland development.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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ABSTRACT: Carbonic anhydrase VI (CA VI), encoded by type A transcripts of the gene Car6, is a secretory product of salivary glands and is found in the enamel pellicle. Because higher caries prevalence is associated with lower salivary concentrations of CA VI in humans, we tested whether CA VI protects enamel surfaces from caries induced by Streptococcus mutans, using Car6(-/-) mice, in which salivary CA VI expression is absent. We detected aberrant Car6 type A transcripts in Car6(-/-) mice, likely targets for nonsense-mediated mRNA decay. Expression of the intracellular stress-induced isoform of CA VI encoded by type B transcripts was restricted to parotid and submandibular glands of wild type mice. The salivary function of Car6(-/-) mice was normal as assessed by the histology and protein/glycoprotein profiles of glands, salivary flow rates and protein/glycoprotein compositions of saliva. Surprisingly, total smooth surface caries and sulcal caries in Car6(-/-) mice were more than 6-fold and 2-fold lower than in wild type mice after infection with S. mutans strain UA159. Recoveries of S. mutans and total microbiota from molars were also lower in Car6(-/-) mice. To explore possible mechanisms for increased caries susceptibility, we found no differences in S. mutans adherence to salivary pellicles, in vitro. Interestingly, higher levels of Lactobacillus murinus and an unidentified Streptococcus species were cultivated from the oral microbiota of Car6(-/-) mice. Collective results suggest salivary CA VI may promote caries by modulating the oral microbiota to favor S. mutans colonization and/or by the enzymatic production of acid within plaque.
Biochimica et Biophysica Acta 09/2011; 1812(12):1567-76. · 4.66 Impact Factor
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ABSTRACT: The recently identified gene Muc19/Smgc encodes two diverse splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19). Muc19 is a member of the large gel-forming mucin family and is an exocrine product of sublingual mucous salivary glands in mice. SMGC is a transiently expressed secretion product of developing rodent submandibular and sublingual glands. Little is known about the expression of Muc19/Smgc gene products in other murine salivary and non-salivary tissues containing the mucous cell phenotype. Muc19 expression was therefore initially assessed by RT-PCR and immunohistochemistry. As a complementary approach, we developed a knockin mouse model, Muc19-EGFP, in which mice express a fusion protein containing the first 69 residues of Muc19 followed by enhanced green fluorescent protein (EGFP) as a marker of Muc19 expression. Results from both approaches are consistent, with preferential Muc19 expression in salivary major and minor mucous glands as well as submucosal glands of the tracheolarynx and bulbourethral glands. Evidence also indicates that individual mucous cells of minor salivary and bulbourethral glands produce another gel-forming mucin in addition to Muc19. We further find tissue expression of full-length Smgc transcripts, which encode for SMGC, and are restricted to neonatal tracheolarynx and all salivary tissues. (J Histochem Cytochem 58:141-156, 2010).
Journal of Histochemistry and Cytochemistry 10/2009; 58(2):141-56. · 2.72 Impact Factor
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ABSTRACT: Muc19/Smgc expresses two splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19), the latter a major exocrine product of differentiated murine sublingual mucous cells. Transcripts for Smgc were detected recently in neonatal sublingual glands, suggesting that SMGC proteins are expressed during initial salivary mucous cell cytodifferentiation. We therefore compared developmental expression of transcripts and translation products of Smgc and Muc19 in sublingual glands. We find abundant expression of SMGC within the initial terminal bulbs, with a subsequent decrease as Muc19 expression increases. During postnatal gland expansion, SMGC is found in presumptive newly formed acinar cells and then persists in putative acinar stem cells. Mucin levels increase 7-fold during the first 3 weeks of life, with little change in transcript levels, whereas between postnatal days 21 and 28, there is a 3-fold increase in Muc19 mRNA and heteronuclear RNA. Our collective results demonstrate the direct transition from SMGC to Muc19 expression during early mucous cell cytodifferentiation and further indicate developmentally regulated changes in Muc19/Smgc transcription, alternative splicing, and translation. These changes in Muc19/Smgc gene expression delineate multiple stages of salivary mucous cell cytodifferentiation and subsequent maturation during embryonic gland development through the first 4 weeks of postnatal life.
Journal of Histochemistry and Cytochemistry 01/2009; 57(4):383-96. · 2.72 Impact Factor
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ABSTRACT: We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.
Biochemical and Biophysical Research Communications 09/2007; 359(3):438-44. · 2.48 Impact Factor
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ABSTRACT: Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca(2+) signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca(2+) homeostasis. To monitor intracellular Ca(2+) concentration ([Ca(2+)](i)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in [Ca(2+)](i) in a physiological Ca(2+)-containing solution, with an EC(50) of 2.5 microM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca(2+)](i), and the P1-receptor agonist adenosine caused only a small increase in [Ca(2+)](i). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca(2+)](i), indicating P2Y receptors were involved in this process. In a Ca(2+)-free bath solution, thapsigargin and ATP induced intracellular Ca(2+) release from an identical pool. Nucleotides caused an increase in [Ca(2+)](i) in the potency order of UTP = ATP > ATP gamma S > ADP > UDP that is best fitted with the P2Y(2) subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca(2+)](i) as did ATP, a small but sustained increase in [Ca(2+)](i) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in [Ca(2+)](i) could be blocked by either nonselective cation channel blockers Gd(3+) or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd(3+) or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca(2+)](i) was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X(1) and P2Y(2) receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca(2+) signaling.
American journal of physiology. Renal physiology 08/2004; 287(2):F204-14. · 3.68 Impact Factor
