Subcloning and regulation of a human intestinal sodium-phosphate cotransporter gene

Abstract

Phosphate plays a critical role in the body as a constituent of bone and tooth for body development, and as a urinary buffer for pH in body acid-base balance regulation. The phosphorus level in blood in human is between 3.0 to 4.5 mg/dl. When the blood phosphate concentration is lower than 2.5 mg/dl, the person develops hypophosphatemia. When the blood phosphate concentration is higher than 4.5 mg/dl, the person develops hyperphosphatemia. It is critically important for the body to control the phosphate level in blood and maintain the phosphate homeostasis. The kidney and the intestine are the important sites to regulate phosphate homeostasis. This dissertation research was to explore the transporter gene(s) involved in the intestinal sodium-dependent phosphate absorption in human and to investigate the role of sodium-phosphate (NaPi) transporters in phosphate homeostatic regulation. The research was performed to test the hypothesis that the sodium-dependent phosphate (NaPi) cotransporter is involved in the phosphate absorption in intestine and various physiological regulators modulate the activity of this transporter. A cDNA encoding a novel human small intestinal Na⁺-P i transporter was isolated from a human intestinal cDNA library. This cDNA encodes a 689 amino acid polypeptide which is different from the renal NaPi cotransporters. This human intestinal NaPi cotransporter gene was mapped to human chromosome 4p15.1--p15.3 by the F̲luorescence I̲n-S̲itu H̲y̲bridization (FISH) method. The human intestinal NaPi cotransporter gene structure was studied by screening a human genomic DNA library. This gene contains 12 exons and 11 introns. There were two transcription initiation sites identified by primer extension. In vivo and in vitro studies showed that the intestinal NaPi cotransporter gene expression is regulated by EGF and vitamin D3. EGF inhibits NaPi-cotransporter gene expression, while vitamin D3 stimulates NaPi-cotransporter gene expression. From these studies, I concluded that the intestinal sodium-dependent phosphate absorption is mainly mediated by intestinal NaPi cotransporters (NaPi-IIb), and this transporter is modulated by various physiological regulators in order to maintain the phosphate homeostasis.