Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter

Abstract

We have studied the involvement of proteolytic pathways in the regulation of the Na/Pi cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, L-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (i) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/Pi cotransport and (ii) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/Pi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the Na/Pi cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.

Figure 1

Inhibitors of lysosomal degradation lead to an accumulation of the type II Na/P_i cotransporter and prevent its PTH-mediated degradation. OK cells were pretreated with leupeptin (100 μg/ml) or methylamine (50 mM) for 30 min. After the pretreatment cells were incubated for 4 h with or without PTH (10⁻⁸ M) in the continued presence or absence of the corresponding lysosomal inhibitor. The effects of the lysosomal inhibitors were investigated by immunodetection of the Na/P_i cotransporter on Western blot (A) as well as by measuring Na/P_i cotransport (B).

Figure 2

Inhibition of proteasomal activity by lactacystin does not prevent the PTH-mediated degradation of the type II Na/P_i cotransporter. OK cells were pretreated with different concentrations of lactacystin for 30 min. After the pretreatment cells were incubated for 4 h with or without PTH (10⁻⁸ M) in the continued presence of the corresponding lactacystin concentration. Of each sample equal amounts of protein were analyzed by SDS-PAGE and immunoblotting.

Figure 3

Subcellular distribution of the type II Na/P_i cotransporter in untreated OK cells and in OK cells treated with PTH and/or leupeptin. OK cells were pretreated with nothing or leupeptin (100 μg/ml) for 30 min. After the pretreatment cells were incubated for 4 h with or without PTH (10⁻⁸ M) in the continued presence or absence of the lysosomal inhibitor. Before homogenization cells were surface-labeled with biotin. After centrifugation the Percoll density gradients were fractionated into three fractions of equal volume: fraction I containing low density membranes, fraction II containing membranes of an intermediate density, and fraction III containing high density membranes. Each of these fractions was analyzed for the activity of the lysosomal enzyme marker β-hexosaminidase, the presence of biotinylated cell-surface membranes, and the expression of the Na/P_i cotransporter. To determine the subcellular distribution of the transporter equal volumes of the gradient fractions were analyzed by SDS-PAGE and immunoblotting. In parallel, equal volumes of the same fractions were dot-blotted and probed with streptavidin–horseradish peroxidase to detect biotinylated cell-surface membranes. Depicted above are the distribution of the β-hexosaminidase, the Na/P_i cotransporter, and the biotinylated cell-surface membranes in untreated cells (A), in cells treated for 4 h with PTH (10⁻⁸ M) (B), in cells treated for 4.5 h with leupeptin (100 μg/ml) (C), and in cells pretreated for 30 min with leupeptin and treated thereafter for 4 h with PTH in the presence of the lysosomal inhibitor (D).

Figure 4

Subcellular localization of the type II Na/P_i cotransporter by confocal microscopy in untreated OK cells and in OK cells treated with PTH and/or leupeptin. OK cells grown to confluency on glass coverslips were pretreated with nothing or leupeptin (100 μg/ml) for 30 min. After the pretreatment cells were incubated for 4 h with or without PTH (10⁻⁸ M) in the continued presence or absence of the lysosomal inhibitor. Depicted above are immunohistochemical double stainings of β-actin (red) and the Na/P_i cotransporter (green) in untreated OK cells (A), in cells treated for 4 h with PTH (10⁻⁸ M) (B), in cells treated for 4.5 h with leupeptin (100 μg/ml) (C), and in cells pretreated for 30 min with leupeptin and treated thereafter for 4 h with PTH in the presence of the lysosomal inhibitor (D).

Figure 5

Requirement of de novo synthesis for the recovery of the Na/P_i cotransport from PTH inhibition. OK cells have been preincubated for 30 min with (B) or without (A) leupeptin (100 μg/ml) followed by an incubation for 2 h with PTH (10⁻⁸ M) in the continued presence (B) or absence (A) of leupeptin. Thereafter cells were washed twice with normal medium and incubated for 4 h with (○) or without (▵) cycloheximide (100 μM) in the continued presence (B) or absence (A) of leupeptin.