J Bone Miner Met 1994, VoI 12 (Suppl 1): $19-$22

E n z y m o l o g i c a l and Cel lu lar M e c h a n i s m s of P a r a t h y r o i d H o r m o n e D e g r a d a t i o n by the K i d n e y

Toru Yamaguchi, Masaaki Fukase, Toshitsugu Sugimoto, Kazuo Chihara Third Division, Department of Medicine, Kobe University School of Medicine, Kobe, Japan

Abstract"

Parathyroid hormone (PTH) is known to be degraded in the kidney at both the luminal microvilli and the basolateral surfaces of the proximal renal tubules in vivo.

We have recently isolated a PTH-degrading enzyme from the microvillar membranes of rat kidney. The NH~-terminal amino acid sequence of the purified enzyme was identical to meprin (EC 3, 4, 24,18). Both the microvillar membrane preparation and purified enzyme generated PTH cleavage peptides that have similar sequences. These results clearly show that the PTH-degrading microvillar enzyme is meprin. We also have investigated the cellular mechanisms of PTH-degradation at the basolateral surfaces using the cultured cell line from the proximal renal tubules, opossum kidney (OK) cells, which have a functional PTH receptor but lack luminal microvilli. The results suggest that this process is largely dependent on a receptor-mediated endo- cytosis and subsequent lysosomal hydrolysis and partially on a hydrolytic activity by a chymotrypsin-like endopeptidase in the membranes. The experiments using phorbol esters suggest that the latter activity might be augmented by protein kinase C activation.

Key words: parathyroid, kidney, degradation, meprin, protein kinase C

To explain the heterogeneity of circulating forms of parathyroid hormone (PTH), degrading activities on the hormone have been demonstrated in the parathy- roid gland, liver and kidney [1]. Intracellular PTH metabolism in the parathyroid gland participates in secretory regulation. Large amounts of PTH are pro- cessed to C-terminal fragments before exocytosis into the blood, probably through cleavage of PTH at the 23-24 bond by nonlysosomal enzymes [2]. Thus, the parathyroid gland secretes not only intact PTH-(1-84) but also biologically inactive its C-terminal fragments [3, 4]. Since the amounts of PTH fragments secreted from the organ increase under hypercalcemic condi- tions, with concomitant decrease in those of intact

PTH [3,41, the secretion of PTH fragments seems to be positively controlled by serum calcium concentra- tion. Other two organs, the liver and kidney, are involved in the peripheral metabolism of PTH. From the animal study in vivo [5 ] , Martin et al. EI~ proposed that intact PTH was selectively uptaken and cleaved by the liver into equal amounts of N-terminal and C-terminal fragments that were not further metabolized by the liver, although controversies still exist about this issue [6]. Enzymologieally, Diment et al. [7] suggest that cathepsin D in Kupffer cells plays a main role in PTH degradation by the liver. They have reported that rabbit macrophages, the presumable precursors of Kupffer cells, selectively

Please address all reprint requests to: Toru Yarnaguchi, M.D. Third Division, Department of Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan

S 20 Journal of Bone and Mineral Metabolism Vol, 12 (Suppl 1), 1994

cleaved intact PTH into equal amounts of the bioactive N-terminal fragment, PTH-(1-34), and the counter- part C-terminal one, by hydrolytic activity of cathe- psin D in endosomes without delivery to lysosomes, and that the generated fragments returned to the extracellular medium. In contrast to the liver, which may have a potential to produce a bioactive N-terminal PTH fragment, the kidney is thought to play a role only in removing PTH from the circulation and abolishing its biological activity in vivo, by degrading the hormone at the luminal microvillar surfaces of the proximal renal tubules after their glomerular f i l trat ion [1,6, 8,9]. A small part of intact PTH and its bioactive N-terminal fragments is also known to be degraded at the basolateral surfaces of the proximal renal tubules by uptake from the peritubular capillaries [1,91. Although these findings have been clarified mainly by animal studies in vivo and by isolated perfused kidneys, studies using biochemical methods or cultured renal cells have been little performed to date. Hence, a microvillar PTH-degrading enzyme in the luminal microvillar surfaces or the cellular mechanisms of PTH uptake by the basolateral surfaces have remained unclear.

Enzymological mechanism of PTH degradation at the microvillar membranes of the proximal renal tubules To clarify the biochemical property of a PTH-degrad- ing enzyme in the luminal microvilli of the proximal reflal renal tubules, we isolated and characterized a metalloendopeptidase hydrolyzing PTH from the microvillar membranes of ra t kidney [10]. The puri- fied enzyme hydrolyzed human PTH-(1-84) , atr ial natriuretic peptide ( A N P ) , insulin B-chain and lyzozyme at the peptide bonds involving a hydrophilic amino acid [10, 11]. The purifed enzyme had apparent molecular masses of 88 kDa and 245 kDa on sodium dodecyl su l fa te /polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respec- tively, showing that it has an oligomeric structure. Further immunological characterization using an anti- body against the purified enzyme [12] revealed that the enzyme was a glycoprotein, with a membranebound anchoring domain, and was located on the microvilli of the proximal renal tubules and intestine. These structure and distribution of the purified PTH- degrading enzyme are quite similar to another kidney microvillar enzyme, meprin [13-17]. Hence, we further investigated whether the purified PTN-degrading enzyme was structurally identical to meprin, by determining its N-terminal amino acid se- quence with an automated gas-phase protein sequencer. The purified enzyme has the N-terminal amino acid sequence NALRDPXXRWKPXIPYILADNLXXNAK,

which is identical to the a subunit of ra t meprin [18]. Thus, the purified PTH-degrading enzyme is shown to be meprin itself. We examined whether the purified enzyme, or ra t meprin, was involved in PTH degradation by the microvillar membranes of ra t kidney as an integral membrane protein [18]. Reverse-phase HPLC analysis revealed that both meprin and the microvillar membranes of ra t kidney readily hydrolyzed human PTH- (39-84) and human PTH- (39-68) into some fragments. The PTH-degrading activity in the micro- villar membranes was not inhibited by addition of phosphoramidon, a potent inhibitor of neutral endopeptidase 24.11 (NEP) E19,20], indicating that NEP is not involved in PTH degradation by the microvillar membranes of ra t kidney. This phenome- non shows marked contrast to the renal metabolism of ANP and endothelin-1, in which NEP is known to play an almost exclusive role [21,22]. The N-terminal amino acid sequences of the degradation products by meprin and by the microvillar membranes of ra t kid- ney were determined with a protein sequencer. Both meprin and the microvillar membranes of ra t kidney cleaved PTH between hydrophilic amino acid residues, which sites are compatible with the hydrolytic pro- perty of meprin but not of NEP that has a preference for hydrophobic amino acids. The amino acid se- quences of three of four major HPLC peaks of metabo- lites by the microvillar membranes of ra t kidney completely corresponded to the metabolites produced by meprin, indicating that most of the PTH-degrading

activity in the microvillar membranes is ascribed to meprin integrated in the membranes. In conclusion,

the purified microvillar endopeptidase, or meprin, is thought to be predominantly involved in PTH degrada-

tion by the microvillar membranes of rat kidney as

an integral membrane protein in vi tro. Although meprin's role in vivo has remained unclear in contrast to its well-documented biochemical charecters, this finding first suggests the physiological importance of meprin in PTH degradation by the kidney.

Cellular mechanisms of PTH degradation at the baso- lateral surfaces of the proximal renal tubules We used an opossum kidney (OK) cell line to elucidate the cellular mechanisms of PTH degradation at the basolateral site of the proximal renal tubules by up- take from peritubular capillaries. Although OK cells are a good cultured cell model for the proximal renal tubules in that they have both PTH receptors coupled to a cAMP production and sodium-dependent phos- phate t ransport E23,24], these ceils are known to lack normal microvillar structure at the luminal surfaces and thus are not completely equal to the well- differentiated proximal renal tubular cells [25]. AI-

Enzymological and Cellular Mechanisms of Renal PTH Degradation S 21

ternatively, OK cells are suitable for studying PTH degradation at the basolateral surface specifically, because the effect of luminal microvillar hydrolytic activities is negligible. Indeed, our study showed that C-terminus or mid-portion of PTH, hPTH-(39-84) or hPTH-(39-68), which are known to be hydrolyzed solely at the luminal microvillar membranes in animal studies [1,6], were scarcely degraded by the monolayer cultures" of OK cells [26]. In contrast, about 70 % of hPTH-(1-84) added in the media was readily hydrolyzed by these cells via a receptor-mediated endocytosis and subsequent lysosomal hydrolysis at the basolateral surfaces. This lysosomal process seems quantitatively important in PTH degradation at the basolateral surface of the proximal renal tubules. In addition, we indicated that OK cells degraded about 5 ~ of hPTH-(1-84) added in the media through a receptor-mediated nonlysosomal pathway [27] . Since this process selectively cleaved PTH at several peptide bonds, and was characteristically inhibited by chymostatin, an inhibitor of chymotrypsin-like proteases, the participation of a chymotrypsin-like endopeptidase was suggested in the process. The simi- lar nonlysosomal PTH degradation was also observed in an osteoblast-like UMR-106 cell line [28, 29], which has PTH receptors coupled to dual signal transduction systems (protein kinase C and cAMP) like an OK cell line [301. In both cell lines, addition of activators of protein kinase C such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-glycerol (OAG) into the media enhanced this degradation pathway [31,321. Hence, this nonlysosomal PTH degradation, to which the receptor binding of the hormone is pre- requisite for its occurrence, seems to be modulated by protein kinase C, one of the dual signal pathways coupled to PTH receptors, in a feedback fashion.

Physiological significance of PTH degradation in vivo The physiological meaning of the heterogeneity of PTH molecules in the circulation is not fully under- stood at present. Martin et al. [33] reported that the perfused canine bone selectively uptook PTH-(1-34) with concomitant cAMP production but not PTH-(1- 84), and suggested the importance of the limited cleav- age of PTH-(1-84) by the liver, in that it produced PTH-(1-34), the only bioactive PTH peptide for the bone. Although controversies still exist about this issue, Sugimoto et al. [34] also confirmed this phe- nomenon with a rat bone perfusion system. Recently, Murray et al. [351 demonstrated that hPTH-(53-84) stumulated dexamethasone-indueed alkaline phos- phatase activity in osteoblastic ROS 17/2.8 cells, suggesting that C-terminal PTH fragments might have some biological activities in osteoblasts. More-

over, Kaji et al. [36] demonstrated that C-terminal PTH fragments possessed the ability to stimulate osteoclast-like cell formation as well as bone-resorp- tion, suggesting the physiological importance of the C-terminal fragments in osteoclastic function. Both studies suggest the biological action of the C-terminal fragments of PTH on bone cells, and seem to require further studies on the relation between the accumula- tion of C-terminal fragments of PTH and bone lesions, both of which are typically observed in renal osteo- dystrophy in hemodialysis patients.

Acknowledgments

This study was in part supported by a Grant-in-Aid for Encouragement of Young Scientists (NO. 05770098) from the Japanese Ministry of Education, Science and Culture to T.Y. and a SRF grant for Biomedical Research to M.F.

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