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

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.