Circulating PTH molecular forms: What we know and what we don t

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1 & 6 International Society of Nephrology irculating PTH molecular forms: What we know and what we don t P D Amour 1,2 1 Parathyroid Physiology Laboratory, entre de recherche, entre hospitalier de l Université de Montréal (HUM) Hôpital Saint-Luc, Montréal, Québec, anada and 2 Université de Montréal, Montréal, Québec, anada irculating parathyroid hormone (PTH) molecular forms have been identified by three generations of PTH assays after gel chromatography or high-performance liquid chromatography fractionation of serum. arboxyl-terminal () fragments missing the amino-terminal (N) structure of PTH(1 84) were identified first. They represent 8% of circulating PTH in normal individuals and up to 95% in renal failure patients. They are regulated by calcium (a) slightly differently than PTH(1 84), occurring in a relatively smaller proportion relative to the latter in hypocalcemia but in a much larger proportion in hypercalcemia. Synthetic -PTH fragments do not bind to the PTH/PTHrP type I receptor and are not implicated in the classical biological effect of PTH(1 84). They bind to a different -PTH receptor and exert biological actions on bone that are opposite to those of PTH(1 84). The integrity of the distal -structure appears to be important for these biological effects, and it is uncertain if all -PTH fragments are intact up to position 84. A second category of -PTH fragment has a partially preserved N-structure. They are called non-(1 84) PTH or N-truncated fragments. They react in Intact (I)-PTH assays but not in PTH assays with a 1 4 epitope. They are acutely regulated by a 2 þ concentration. They also exert similar hypocalcemic and antiresorptive effects but have 1-fold greater affinity for the -PTH receptor compared to other -PTH fragments. Even if they represent only 1% of all -PTH fragments, they could be as relevant biologically. An N form of PTH other than PTH(1 84) has been identified in the circulation. It reacts very well in PTH assays with a 1 4 epitope but poorly in I-PTH assay with a epitope. It is oversecreted in severe primary and secondary hyperparathyroidism and in parathyroid cancers. Its biological activity is still unknown. Overall, these studies suggest that PTH(1 84) and -PTH fragments are regulated differently to exert opposite biological effects on bone via two different receptors. This may serve to control bone turnover and a concentration more efficiently. Kidney International (6) 7, S29 S33. doi:1.138/sj.ki.599 orrespondence: P D Amour, Parathyroid Physiology Laboratory, entre de recherche, entre hospitalier de l Université de Montréal (HUM) Hôpital Saint-Luc, 264, boul. René-Lévesque est, Montréal, Québec, anada H2X 1P1. rechcalcium.chum@ssss.gouv.qc.ca KEYWORDS: parathyroid hormone (PTH); PTH(7 84); N-PTH; -PTH fragments; renal failure; calcium THE IRULATING PARATHYROID HORMONE (PTH) PARADOX PTH(1 84) is the main synthetic product of parathyroid cells in most species. 1 It exerts its classical biological effects on bone and kidney through its first 34 amino acids, that is, its amino-terminal (N) structure. 2 The latter binds to the type I PTH/PTHrP receptor and activates both the protein kinase A and pathways in target tissues. 3 The carboxyl-terminal () structure of PTH(1 84), the last 5 amino acids, has no known direct influence on this receptor. 4,5 This biological reality poorly reflects the composition of circulating PTH in humans and animals, as it is mainly comprised of -PTH fragments, 6,7 constituting a major paradox that is just beginning to be resolved. The quantity of -PTH fragments relative to that of PTH(1 84) is both acutely and chronically well-regulated, which has biological implications. 8 These have been difficult to uncover because a less well-characterized, not yet cloned receptor appears to be involved. 9 The central theme of our presentation is what we know and do not know about circulating PTH molecular forms and their biological effects through two different PTH receptors. IRULATING PTH MOLEULAR FORMS AND PTH ASSAYS Two main findings, that PTH(1 84) biological effects were only dependent on its N-structure 2 and that circulating PTH was mainly composed of -PTH fragments, 6,7 have influenced the evolution of PTH assays. They were believed to limit the capacity of PTH measurements to reflect the true biological activity of the hormone with radio-immunoassays (RIAs) having epitopes in the -structure of the hormone, such as mid- and -terminal PTH RIAs. This was particularly true in terminal renal failure patients, where these fragments accumulate and represent more than 95% of the hormone in the circulation, 1 complicating correlation analysis with any measurements reflecting PTH(1 84) biological effects. 11 This has dictated the development of a second generation of PTH assay, Intact (I)-PTH assay, which uses solid-phase capture antibodies purified by affinity chromatography against hpth(39 84) and revealing antibodies purified against Kidney International (6) 7, S29 S33 S29

2 P D Amour: irculating PTH hpth(1 34). Most of the latter have their main epitope in region of the PTH(1 34) structure, early epitopes like and being more frequent than distal epitope These assays do not detect mid- or -PTH fragments missing a N-structure, and were believed to react only with hpth(1 84). They had a major impact in the PTH field both clinically and experimentally, allowing the comparison of quantitative results between laboratories for the first time. 13,14 They were eventually demonstrated to react with -PTH fragments having a partially preserved N-structure, also called non-(1 84) PTH fragments or N-truncated -fragments, by my laboratory. 1 These fragments accumulate and represent up to 45% of the immunoreactivity in renal failure patients, but only % in normal individuals. 1 Since this was believed to impair their capacity to appreciate hpth(1 84) biological effects in vivo, particularly in terminal renal failure patients, a third generation of PTH assays was developed. It uses the same capture antibodies as second-generation assays, but revealing antibodies with a 1 4 epitope. 16,17 This eliminates the immunoreactivity associated with hpth(7 84) or non-(1 84) PTH fragments in the second generation of PTH assays. 16,17 These assays were believed to be specific for hpth(1 84) until we again demonstrated that they also react with an N-form of PTH distinct from hpth(1 84) on serum PTH high-performance liquid chromatography (HPL) profiles obtained in normal individuals and in patients with primary or secondary hyperparathyroidism. 18 This molecular form of PTH is poorly reactive in I-PTH assays with early epitopes covering region but fully reactive in I-PTH assays with a distal epitope, suggesting some modification in region. 12,18 A representative illustration of PTH molecular forms detected by three generations of PTH assays is presented in Figure 1. -PTH FRAGMENTS: ORIGIN REGULATION -PTH fragments, with or without a partially preserved N-structure, are secreted by parathyroid glands in a calciumdependent manner 1,19 and are produced during the peripheral metabolism of PTH(1 84) in liver Kuppfer cells. 24 They undergo renal clearance, which explains their accumulation in renal failure patients. 27 -PTH fragments are acutely regulated in the circulation by ionized calcium (a 2 þ ) concentration. 1,28,29 This regulation operates within the normal range of a 2 þ values in humans. While stimulating PTH secretion, hypocalcemia favors the output of PTH(1 84) over that of -PTH fragments, decreasing the -PTH fragments/pth(1 84) ratio to its lowest value. 1,28 While inhibiting PTH secretion, hypercalcemia favors the output of -PTH fragments over that of PTH(1 84), increasing the -PTH fragments/ PTH(1 84) ratio to its highest value. 1,28 This applies to both types of -PTH fragments. 1, Mid-and non RF N -fragments (1-84) N (1-84) Mid -PTH epitope (49-53) AN (%) 5% Normal Mid 95% Renal failure 2% Mid 98% -PTH 1 epitope (64-69) AN (%) 18% 82% 5% 95% T-PTH A-PTH epitope (12-18) epitope (1-4) AN (%) AN (%) 8% 93 PTH (1-84) N % 7% 55% 85% Other PTH N 45% % Figure 1 omposition of circulating PTH in a normal individual ( ) and a terminal renal failure patient ( ) after high-pressure liquid chromatography (HPL) separation of serum and analysis of fractions with PTH assays of different specificities. Mid- and -PTH assays are first-generation PTH radioimmunoassays with epitopes in the -structure (last 5 amino acids) of human (h) PTH(1 84). The total (T)-PTH assay is a second-generation PTH assay, similar to other intact PTH assays, with an epitope in the region of the N structure of hpth(1 84). Finally, the cyclase-activating (A) PTH assay is a third-generation assay with a 1 4 epitope. With each generation, PTH assays have become more and more specific (but not exclusive) for hpth(1 84), the bioactive form of the hormone on the type I PTH/PTHrP receptor. S Kidney International (6) 7, S29 S33

3 P D Amour: irculating PTH Apart from the acute regulation of circulating PTH molecular forms, further chronic regulation can be demonstrated based on PTH(1 84) needs. When more PTH is needed to maintain a 2 þ concentration, as in evolving secondary hyperparathyroidism due to vitamin D deficiency, 31 renal failure 1 or half parathyroidectomy, 32 more PTH(1 84) than -PTH fragments is secreted at all a 2 þ concentrations, resulting in a lower -PTH fragments/ PTH(1 84) ratio than initially present. 1,31,32 The same is true when less PTH(1 84) is needed, as in chronic nonparathyroid hypercalcemia, 33 during vitamin D replacement therapy in vitamin D-deficient animals 34 and during 1,(OH) 2 D administration in dogs 32 ; less PTH(1 84) than -PTH fragments is secreted at all a 2 þ concentrations, culminating in a higher -PTH fragments/pth(1 84) ratio than initially present This tight regulation of circulating -PTH fragments has some kind of biological implication. -PTH FRAGMENTS: STRUTURE AND BIOLOGIAL ATIVITY -PTH fragments produced during the peripheral metabolism of 1 I-bPTH(1 84) in dogs and rats 36 start their N-structure at positions 34, 37, 41 and 43 of the bpth structure. Porcine parathyroid cells also secrete internally labelled -PTH fragments starting at positions 34 and The integrity of the -terminal end of these fragments remains an open issue. -PTH RIAs detect hpth(53 83) as well as hpth(53 84), and we do not know how many amino acids must be removed from the -terminal end to impair immunoreactivity in these assays. This is a practical issue, because removing glutamine in position 84 can impair biological activity 38 or greatly reduce affinity for the -PTH receptor. 39 We have studied the N-structure of non-(1 84) PTH fragments labelled internally with S-methionine and secreted by parathyroid cells from patients with primary or secondary hyperparathyroidism. 4 The N-structure of these fragments started at positions 4, 7, 8, 1 and of the hpth(1 84) structure, positions 7 and being dominant. We have also studied the -terminal end of these fragments, using an antibody provided by Immutopics International, which detected hpth(53 84) better than hpth(53 83). omparing the recovery of non(1 84) PTH fragments obtained with this assay to that of an I-PTH assay in HPL profiles, we could conclude that not all non-(1 84) PTH fragments had an intact -terminal end structure. 4 More studies with better -terminal end antibodies are required to confirm these findings. -PTH fragments, with or without a partially preserved N-structure, do not interact with the type I PTH/PTHrP receptor 4,5,41,42 nor do they directly influence camp or the intracellular a 2 þ responses 44 to hpth(1 34) or hpth(1 84). They interact with a less well-characterized -PTH receptor present in chicken renal plasma membranes, 45 in osteoblastic rat osteosarcoma cells 38,41,46,47 and in other cell lines. 47 The presence of a large number of -PTH receptors in osteocyte-like cells, obtained from the bone of mice with type I PTH/PTHrP gene deletion, is particularly revealing. 48 These cells have been used to demonstrate the affinity of various synthetic -PTH fragments for the -PTH receptor with a tracer specific to the receptor, 1 I[tyr 34 ] hpth(19 84). 39 -PTH fragments larger than hpth(24 84) had an affinity equal to hpth(1 84) for this receptor and 1 times higher than that of fragments smaller than hpth(28 84). Removal of position 84 also greatly reduced the affinity of hpth(53 83) compared to that of hpth(53 84), and fragments smaller than hpth(53 84) did not displace the tracer. 39 In ROS 17/2.8 clonal cells, hpth(69 84) was the smallest structure able to interact with the -PTH receptor, 38 and in chondrocytes, region 73 76, present in 57 76, was found necessary to stimulate an increase of intracellular a 2 þ. 49 This suggests that different regions of the -structure of hpth(1 84) may be involved in binding to and activation of the -PTH receptor. Several biological effects, which are often opposite to those of hpth(1 34) on the type I PTH/PTHrP receptor, have been associated with synthetic -PTH fragments in vitro and in vivo. 9 In vitro, synthetic -PTH fragments missing an N-structure stimulate alkaline phosphatase activity and osteocalcin mrna in ROS 17/2.8 and SaOS-2 clonal cells, 38,5 53 type 1 Procollagen and insulin-like growth factor-binding protein-5 mrna expression in UMR-16 cells, 54 basal osteoclastic-like cell formation and osteoclastic activity, 44 alkaline phosphatase activity in mouse embryo tooth germ 55 and collagen expression in chondrocytes. 56 They also inhibit [ 3 H]thymidine incorporation into the condylar cartilage of neonatal mice. 57 hpth(7 84), a -PTH fragment with a partially preserved N-structure, is a potent inhibitor of bone resorption induced by a variety of substances. 58 It also inhibits vitamin D-induced osteoblastic cell differentiation from bone marrow, as does hpth(39 84), but at a 1 times higher molar concentration. 58 Synthetic -PTH fragments also promote osteocyte apoptosis. 39,48 Short-term in vivo studies have been performed in parathyroidectomized rats. 41,43 hpth(7 84) alone decreased calcium concentration in these animals, as did a mixture of synthetic -PTH fragments at 1 times the concentration of the former. 41,43 Both preparations also substantially reduced the calcemic response to hpth(1 34), but only hpth(7 84) the calcemic response to hpth(1 84). 41,43 These preparations also caused a decrease of serum phosphate 41 and of the phosphaturia induced by hpth(1 84), 43 suggesting the input of calcium and phosphate into bone. A more chronic experiment performed in PTX uremic animals 59 did not detect a hypocalcemic effect of hpth(7 84) over 3 weeks, but indicated that hpth(7 84) inhibited the bone turnover rate induced by hpth(1 84) alone. N-FORMS OF IRULATING PTH The existence of N-forms of PTH distinct from PTH(1 84) has always been a very controversial issue. These molecular forms are not produced during the peripheral metabolism of S internally labelled PTH(1 84). 6 Using third-generation PTH assays and a more performant HPL gradient, we Kidney International (6) 7, S29 S33 S31

4 P D Amour: irculating PTH recently demonstrated the existence of an N-form of PTH, distinct from hpth(1 84) and slightly more hydrophilic in normal individuals and in patients with primary and secondary hyperparathyroidism. 18 This molecular form of PTH reacts poorly in I-PTH assays having early or epitopes, but normally in assays with a epitope, suggesting a modification in region. 12,18,4 The only post-translational modification ever described for PTH(1 84) is phosphorylation in the N-terminal region. 61 This could explain the poor immunoreactivity of PTH assays having epitopes covering region. N-PTH represents 4 8% of the immunoreactivity detected by a third-generation PTH assay in normal individuals and up to % in renal failure patients. 12,18 This molecular form of PTH can be overproduced in severe primary 18,62 or secondary 63 hyperparathyroidism, as well as in parathyroid cancer. 64 Its role in PTH physiology as well as its biological activity remain obscure at this point. Further studies will be required to investigate these issues. PTH BIOLOGY: THE NEW EMERGING VERSION Better knowledge of circulating PTH molecular forms and of their biological effects through two different PTH receptors has changed our conception of the regulation of calcium metabolism and bone turnover. From a version which allowed only positive input of the N-structure of PTH(1 84) on calcium and bone turnover through the type I PTH/PTHrP receptor, we have evolved toward a new version that also includes a second negative input component which exerts opposite effects through a second -terminal receptor. What we measure represents the summation of these two components. Several levels of interaction between N-forms Type I Ι PTH/PTHrP P receptor Biological(+) effects hpth(1-84) Self, additive, opposite biological effects -PTH fragments -PTH H receptor or Biological( ) effects Figure 2 The biological activity of PTH can no longer be limited to the interaction of hpth(1 84) with the type I PTH/PTHrP receptor. First, hpth(1 84) is a minor form of circulating PTH, and other molecular forms, more representative of the structure of hpth(1 84) and abundant in the circulation, appear to exert biological effects often opposite to those of hpth(1 84) through a different, less well-characterized, -PTH receptor. How these receptors talk to each other is just beginning to be appreciated and constitutes a new field of research. It is likely that new levels of calcium and bone turnover regulation will come out of this research. these two systems are likely and will need to be revealed in future studies (Figure 2). AKNOWLEDGMENTS We thank Manon Livernois for typing this manuscript and Ovid Da Silva for editing it. Studies performed in the laboratory of Dr Pierre D Amour were supported in part by grants from the Medical Research ouncil of anada (MA-7643), the anadian Institutes for Health Research (MOP-7643) and NPS Pharmaceuticals. REFERENES 1. Kronenberg HM, Bringhurst RF, Segre GV, Potts Jr JT. Parathyroid hormone. Biosynthesis and metabolism. In: Bilezikian J, Marcus R, Levine MA (eds). The Parathyroids. Academic Press:New York, 1, pp Tregear GW, Van Rietschoten J, Greene E et al. Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity. Endocrinology 1973; 93: Abou-Samra AB, Jüppner H, Force T et al. 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A molecular form of PTH distinct from PTH(1 84) is produced in parathyroid carcinoma. J Bone Miner Res 5; 19: S327 (abstract SU514). Kidney International (6) 7, S29 S33 S33