Ribozyme knockdown functionally links a 1,25(OH) ₂ D ₃ membrane binding protein (1,25D ₃ -MARRS) and phosphate uptake in intestinal cells

Abstract

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)2D3 and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D3-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)2D3 binding, but did not affect binding to the nuclear receptor for 1,25(OH)2D3. Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D3-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)2D3 in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D3-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D3-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Intestinal Cell Calcium Uptake and the Targeted Knockout of the 1,25D3-MARRS (Membrane-associated, Rapid Response Steroid-binding) Receptor/PDIA3/Erp57

“…For the steroid hormone 1,25(OH) 2 D 3 , two possible candidates exist: the classical vitamin D receptor (VDR) (10) and the more recently discovered and unrelated 1,25D 3 -MARRS (membrane-associated, rapid response, steroid binding) receptor/PDIA3/ERp57 (11). Wali et al (7) have reported that the VDR is not required for the rapid actions of 1,25(OH) 2 D 3 in mouse osteoblasts, whereas Mizwicki et al (10) have argued that only the VDR is necessary to mediate the rapid actions of 1,25(OH) 2 D 3 , despite our reports that RNAi against the 1,25D 3 -MARRS receptor, as well as preincubation of intestinal cells with our neutralizing antibody to the 1,25D 3 -MARRS receptor, eliminates the rapid actions of 1,25(OH) 2 D 3 on phosphate uptake (11).…”

“…After blocking nonspecific binding sites (15), membranes were incubated with Ab099 against the N terminus of the 1,25D 3 -MARRS receptor at a 1:5000 dilution, overnight. Ab099 has been extensively characterized in previous publications (11,16). The following morning, the blot was washed and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase for 2 h at room temperature.…”

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1αOHase-deficient mice

“…On the other hand, it has been postulated that 1,25(OH) 2 D may act on the basis of rapid, nongenomic mechanisms (9). In this context, a protein of chicken enterocytes that belongs to the superfamily of glucose-regulated and redoxsensitive proteins was recently described as a 1,25(OH) 2 Dbinding protein (20). However, in mouse small intestine, a change in the abundance of the NaP i -IIb protein was not observed 3 h after the onset of a low-P i diet (12), indicating that a rapid nongenomic mechanism may not be involved in the regulation of NaP i -IIb in mouse small intestine.…”

Activation of Bone Morphogenetic Protein Signaling by a Gemini Vitamin D3 Analogue Is Mediated by Ras/Protein Kinase Cα

“…It was suggested that PKCa activation by 1a,25(OH) 2 D 3 was through the 1a,25(OH) 2 D 3 membrane-associated rapid response steroid binding protein (16,20). The classic VDR, which is known to translocate to the nucleus after ligand binding, has been proposed to be associated with caveolae in the plasma membrane and is responsible for the rapid response to vitamin D ligands (15,50).…”

“…The effects of 1a,25(OH) 2 D 3 and its analogues are mainly mediated through the vitamin D receptor (VDR) or through the membrane-associated signaling pathway (1,15). Membrane-associated responses to 1a,25(OH) 2 D 3 [nongenomic, rapid response to 1a,25(OH) 2 D 3 ], where the mechanism is still unclear, is now considered an essential type of action involved in calcium/phosphate transport, activation of protein kinase C (PKC), and/or the mitogen-activated protein kinase (MAPK) cascade (15)(16)(17)(18).…”