Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality

We have studied the involvement of proteolytic pathways in the regulation of the NayPi 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 NayPi 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 NayPi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the NayPi cotransporter is constitutively transported to and degraded within late endosomesylysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomesylysosomes. Molecular mechanisms involved in rapid increases or decreases in membrane transport fall into two categories: alteration in transport activities (i.e., transporter kinetics) of membrane resident proteins and alterations in the number of transport proteins in the plasma membrane (1). Alterations in the number of transport proteins in the plasma membrane may be achieved by exocytic insertion of preexisting transport proteins from an intracellular vesicular pool into the plasma membrane or by endocytic retrieval of transport proteins back into intracellular storage vesicles [‘‘shuttle’’ mechanism (2–4)], thereby ensuring a rapid and reversible regulation of membrane transport. Also the regulation of sodium-dependent reabsorption of phosphate (Pi) in renal proximal tubules involves alterations in the number of NayPi cotransporters (NayPi cotransporter type II) expressed in the apical membrane. Parathyroid hormone (PTH)-induced phosphaturia is related to an endocytic retrieval of type II NayPi cotransporters from the brush border membrane (5). A similar mechanism explains the reduced Pi reabsorption as observed after Pi refeeding of Pi-deprived rats (6). These two phenomena can also be observed in opossum kidney cells [OK cells (7, 8)], a frequently used ‘‘in vitro’’ system to analyze cellular mechanisms involved in the regulation of proximal tubular transport functions (9). In this cell system PTH leads to a timedependent inhibition of apical NayPi cotransport activity (7). Surprisingly, recovery of the NayPi cotransport from PTH inhibition required de novo protein synthesis (10). Furthermore, PTH treatment of OK cells led to a time-dependent decrease of the type II NayPi cotransporter protein detectable on Western blots and to a parallel decrease in the transporterrelated immunofluorescence at the apical surface (9). These findings suggested that PTH leads to the degradation of the type II NayPi cotransporter. In the present study we have examined the possible involvement of proteolytic pathways in the PTH-mediated regulation of this transporter. Two major proteolytic systems involved in different aspects of protein breakdown are existing in mammalian cells, the lysosomal and the nonlysosomal system (11). Degradation of plasma membrane proteins as well as of proteins entering the cell by receptor-mediated endocytosis or by pinocytosis takes place in lysosomes (12–14). An important nonlysosomal proteolytic pathway is the ubiquitin-proteasome pathway (11, 15, 16) in which soluble nuclear and cytoplasmic proteins are degraded. Also integral membrane proteins that fail to fold or oligomerize correctly were found to be degraded by cytoplasmic proteasomes after they have been exported from the endoplasmic reticulum (17–22). In the present report we demonstrate that PTH leads to the endocytosis and degradation of the renal type II NayPi cotransporter via the lysosomal proteolytic pathway and not to its endocytic retrieval into intracellular storage vesicles. EXPERIMENTAL PROCEDURES Materials. The following reagents were purchased from Sigma: ammonium chloride, chloroquine, leupeptin, Lmethionine methyl ester, methylamine. Lactacystin was obtained from Dr. E. J. Corey (Harvard University). Cells. All cell culture supplies were obtained from GIBCOy BRL (Basel, Switzerland). OK cells (clone 3By2) (24) were maintained in DMEMyHam’s F12 medium (1:1) supplemented with 10% fetal calf serumy22 mM NaHCO3y20 mM Hepesy2 mM L-glutamine in a humidified atmosphere of 5% CO2y95% air at 37°C. Cell-Surface Biotinylation. Biotinylation was performed similarly as previously described (23). All the steps were carried out at 4°C. 100-mm cell culture dishes (Corning) were washed 5 times with ice-cold PBS containing 1 mM MgCl2y0.1 mM CaCl2 (PBS-CyM) and gently agitated for 30 min at 4°C. Sulfo-N-hydroxysuccinimide-LC-biotin stock solutions (200 mgyml in dimethyl sulfoxide) were stored frozen at 220°C and thawed just before use. Sulfo-N-hydroxysuccinimide-LCbiotin (Pierce) was diluted to a final concentration of 0.125 mgyml in ice-cold PBS-CyM and used immediately. To each 100-mm dish 15 ml of this sulfo-N-hydroxysuccinimide-biotin solution was added. After 30 min of gentle agitation at 4°C the dishes were washed 5 times with ice-cold PBS. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y951909-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: PTH, parathyroid hormone; OK cells, opossum kidney cells. §To whom reprint requests should be addressed at: Institute of Physiology, University Zürich-Irchel, Winterthurerstr. 190, CH-8057 Zürich, Switzerland. e-mail: biber@physiol.unizh. ch.