Molecular characterizations of type IIb sodium dependent phosphate cotransporter in mouse intestine

Persistent Link:
http://hdl.handle.net/10150/279967
Title:
Molecular characterizations of type IIb sodium dependent phosphate cotransporter in mouse intestine
Author:
Arima, Kayo
Issue Date:
2002
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Inorganic phosphate (Pi) homeostasis is mainly regulated by absorption of dietary Pi in the small intestine and reabsorption of filtered Pi in the kidney. I have characterized the 5 '-flanking region and overall gene structure of the murine type IIb sodium-phosphate cotransporter (Na/Pi-IIb), a major apical Pi transporter. The Na/Pi-IIb cotransporter gene spans more than 18 kb and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity when transfected into in rat intestinal epithelial (RIE-1) cells. Pi requirement during development is much higher than in adult life. In the next set of experiments, I sought to characterize expression of the intestinal Na/Pi-IIb cotransporter during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na/Pi uptake by intestinal brush-border membrane vesicles was highest at 14-days-of-age, lower at 21 days and further reduced at 8 weeks and 8--9 months of age. Na/Pi-IIb mRNA and immunoreactive protein levels in 14-d animals were markedly higher than in older groups. MP treatment significantly decreased Na/Pi uptake, and Na/P i-IIb mRNA and protein expression in 14-d mice. Additionally, the size of the protein was smaller in 14-d mice. Deglycosylation of protein from 14-d and 8-wk old animals with PNGase F reduced the molecular weight to the predicted size. I conclude that intestinal Na/Pi uptake and Na/Pi-IIb expression are highest at 14-d and decrease with age. Furthermore, MP treatment reduced intestinal Na/Pi uptake ∼3-fold in 14-d mice and this reduction correlates with reduced Na/Pi-IIb mRNA and protein expression. I also demonstrate that Na/Pi-IIb is an N-linked glycoprotein and that glycosylation is age-dependent. In conclusion, the mouse intestinal Na/Pi-IIb cotransporter is developmentally regulated at mRNA and protein levels. MP-treatment also reduces mRNA and protein expression during development. The Na/Pi-IIb gene promoter constructs identified in the first study will be a useful tool to investigate the possible transcriptional regulations. Furthermore, studying post-translational regulation including glycosylation will reveal developmental effects on Na/Pi-IIb cotransporter protein. These studies will help to decipher molecular mechanisms of Pi absorption in mammalian small intestine.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Genetics.; Biology, Animal Physiology.; Health Sciences, Nutrition.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Nutritional Sciences
Degree Grantor:
University of Arizona
Advisor:
Jones, John J.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleMolecular characterizations of type IIb sodium dependent phosphate cotransporter in mouse intestineen_US
dc.creatorArima, Kayoen_US
dc.contributor.authorArima, Kayoen_US
dc.date.issued2002en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractInorganic phosphate (Pi) homeostasis is mainly regulated by absorption of dietary Pi in the small intestine and reabsorption of filtered Pi in the kidney. I have characterized the 5 '-flanking region and overall gene structure of the murine type IIb sodium-phosphate cotransporter (Na/Pi-IIb), a major apical Pi transporter. The Na/Pi-IIb cotransporter gene spans more than 18 kb and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity when transfected into in rat intestinal epithelial (RIE-1) cells. Pi requirement during development is much higher than in adult life. In the next set of experiments, I sought to characterize expression of the intestinal Na/Pi-IIb cotransporter during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na/Pi uptake by intestinal brush-border membrane vesicles was highest at 14-days-of-age, lower at 21 days and further reduced at 8 weeks and 8--9 months of age. Na/Pi-IIb mRNA and immunoreactive protein levels in 14-d animals were markedly higher than in older groups. MP treatment significantly decreased Na/Pi uptake, and Na/P i-IIb mRNA and protein expression in 14-d mice. Additionally, the size of the protein was smaller in 14-d mice. Deglycosylation of protein from 14-d and 8-wk old animals with PNGase F reduced the molecular weight to the predicted size. I conclude that intestinal Na/Pi uptake and Na/Pi-IIb expression are highest at 14-d and decrease with age. Furthermore, MP treatment reduced intestinal Na/Pi uptake ∼3-fold in 14-d mice and this reduction correlates with reduced Na/Pi-IIb mRNA and protein expression. I also demonstrate that Na/Pi-IIb is an N-linked glycoprotein and that glycosylation is age-dependent. In conclusion, the mouse intestinal Na/Pi-IIb cotransporter is developmentally regulated at mRNA and protein levels. MP-treatment also reduces mRNA and protein expression during development. The Na/Pi-IIb gene promoter constructs identified in the first study will be a useful tool to investigate the possible transcriptional regulations. Furthermore, studying post-translational regulation including glycosylation will reveal developmental effects on Na/Pi-IIb cotransporter protein. These studies will help to decipher molecular mechanisms of Pi absorption in mammalian small intestine.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Genetics.en_US
dc.subjectBiology, Animal Physiology.en_US
dc.subjectHealth Sciences, Nutrition.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineNutritional Sciencesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorJones, John J.en_US
dc.identifier.proquest3050329en_US
dc.identifier.bibrecord.b42727893en_US
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