Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-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)(2)D(3) 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 1,25D(3)-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)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-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)(2)D(3) 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 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Fig. 1.

(A) Sequence of the full-length cDNA and translated polypeptide. Amino acids highlighted in blue correspond to the predicted signal peptide, and amino acids highlighted in yellow correspond to the N terminus of the purified protein recognized by antibody 099 and used for back translation and database searching. Consensus thioredoxin motifs are highlighted in purple. The transcript contains a long 3′ UTR after the KEDL termination sequence. (B) Schematic of targeted portion of MARRS transcript and site of ribozyme cleavage by MARRS 6 ribozyme.

Fig. 2.

The 1,25D₃-MARRS protein mediates phosphate uptake and PKC activation. Isolated intestinal epithelial cells were cultured overnight without serum. The following day (A), media were replaced with GBSS/0.1% BSA without or with Ab 099 (1:500 dilution) and incubated for 5 min (23°C). Each dish then received 1 ml of GBSS containing 4 μCi of H₃³²PO₄ and vehicle (Con and cells pretreated with antibody, AbCon) or 130 pM 1,25(OH)₂D₃ (final concentration; 1,25D₃ and cells pretreated with antibody, Ab1,25D₃) and incubated for an additional 7 min. (B-E) Cells were either not transfected (No Rx) or transfected with MARRS 6 or MARRS 8 ribozymes for 5 h before addition of serum and were assayed 22 h later. (B) Cells were incubated for 7 min for ³²P uptake as in A but without the antibody preincubation. (C) Cells were incubated for 1 min with or without hormone in GBSS and then collected for PKC analyses. (D) Cell lysates were incubated with [³H]1,25(OH)₂D₃ in the absence or presence of excess unlabeled hormone. Bound and free steroid were separated by perchloric acid precipitation to determine 1,25D₃-MARRS-protein-specific binding (D) or by hydroxylapatite to determine specific VDR binding (Inset). (E) Cell lysates (30 μg per lane) were separated on SDS/PAGE and blotted onto poly(vinylidene difluoride) membranes, and Western blot analyses were performed with Ab 099 (against the N terminus of the 1,25D₃-MARRS protein) as primary antibody. Values are presented as mean ± SEM for 6-11 independent samples. ^*, P < 0.02, relative to corresponding controls.

Fig. 3.

Effect of age and gender on the rapid, 1,25(OH)₂D₃-mediated stimulation of phosphate transport in perfused duodenal loops. Chickens were raised on a normal, vitamin D-replete diet for 7, 14, or 28 weeks before experimentation. Arterial perfusion with GBSS containing 0.125% (wt/vol) BSA and the vehicle ethanol [0.005% (vol/vol), final concentration] proceeded for a 20-min basal period, and samples were collected during the latter 10 min. At T = 0, arterial perfusion either continued with control media (open circles) or 130 pM 1,25(OH)₂D₃ (filled circles). The lumen was perfused with GBSS lacking bicarbonate and containing 2 μCi/ml of H₃³²PO₄. Transport was measured by the amount of radioactivity appearing in the venous effluent. Results are presented as mean ± SEM (or range) for 7-week-old males (A) (n = 4 controls and 4 treated), 14-week-old males (B) (n = 5 controls and 3 treated), 28-week-old males (C) (n = 2 controls and 4 treated), 7-week-old females (D)(n = 2 controls and 5 treated), 14-week-old females (E)(n = 3 controls and 4 treated), and 28-week-old females (F) (n = 2 controls and 5 treated). ^*, P < 0.05, relative to corresponding controls for times indicated by arrows.

Fig. 4.

Effect of age and gender on mRNA levels for 1,25D₃-MARRS. Duodenal mucosae were collected from each of the indicated age groups and frozen in liquid nitrogen. Total RNA was prepared with the TRIzol reagent, and 1 μg was used for RT-PCR with primers specific for 1,25D₃-MARRS protein or GAPDH. (Upper) Males. (Lower) Females. The ratios for MARRS/GAPDH are provide underneath each panel.