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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0243
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5774-5778
Copyright © 2005 by The Endocrine Society

Direct in Vitro Evidence of Extracellular Ca2+-Induced Amino-Terminal Truncation of Human Parathyroid Hormone (1–84) by Human Parathyroid Cells

Takehisa Kawata, Yasuo Imanishi, Keisuke Kobayashi, Naoyoshi Onoda, Yoshiaki Takemoto, Hideki Tahara, Senji Okuno, Eiji Ishimura, Takami Miki, Tetsuro Ishikawa, Masaaki Inaba and Yoshiki Nishizawa

Departments of Metabolism, Endocrinology, and Molecular Medicine (T.K., Y.I., K.K., H.T., M.I., Y.N.), Oncology (N.O.), Urology (Y.T.), Nephrology (E.I.), Geriatrics and Neurology (T.M.), and Surgical Oncology (T.I.), Osaka City University Graduate School of Medicine, Osaka 545-8585; and Shirasagi Hospital (S.O.), Osaka 546-0002, Japan

Address all correspondence and requests for reprints to: Yasuo Imanishi, M.D., Ph.D., Department of Metabolism, Endocrinology, and Molecular Medicine, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: imanishi{at}med.osaka-cu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Although serum calcium (Ca2+) concentration regulates the generation of amino-terminally (N-terminally) truncated forms of human PTH (hPTH) degraded from (1–84)hPTH, no studies have yet reported whether the parathyroid gland itself is responsible for this process.

Objective: Our objective was to determine the site of N-terminal truncation and its roles in PTH metabolism in parathyroid cells in vitro.

Methods: The effect of extracellular Ca2+ concentration was examined on N-terminal truncation in primary cultured parathyroid cells. The parathyroid glands were obtained from the patients with primary and uremia-associated secondary hyperparathyroidisms who underwent therapeutic parathyroidectomies.

Results: The N-terminally truncated fragments were detectable with commercially available intact PTH (I-PTH) assays, but not with the bio-intact PTH (Bio-PTH) assay, which detected only the (1–84)hPTH. HPLC revealed that generation of N-terminally truncated fragments detectable by I-PTH increased with extracellular Ca2+ concentration. Suppression of PTH secretion by increasing the extracellular Ca2+ concentration was more evident with the Bio-PTH assay than with the I-PTH assay for both cultured parathyroid cells prepared from parathyroid adenomas and uremia-associated secondary hyperparathyroidism. The Bio-PTH/I-PTH ratio, which is the ratio of (1–84)hPTH to the sum of (1–84)hPTH and N-terminally truncated fragments, decreased in response to increases in extracellular Ca2+.

Conclusions: These findings suggest that the N-terminal truncation is regulated by extracellular Ca2+ concentration and works to suppress the generation of (1–84)hPTH in parathyroid cells.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE SECRETION OF PTH is tightly regulated by extracellular calcium (Ca2+) levels via a calcium-sensing receptor (CaR) (1). The regulation includes a multistep process, involving PTH gene expression (2), PTH mRNA stability (3), and processing steps with intracellular degradation of PTH molecules (4). Extracellular Ca2+ also regulates parathyroid cellular proliferation (5). Two major circulating C-terminal PTH (C-PTH) fragments result from peripheral cleavage of PTH between residues 33 and 34 or between residues 36 and 37 (6, 7). Hypercalcemia inhibits the secretion of human PTH (1–84) [(1–84)hPTH] from the parathyroid gland to a greater extent than secretion of C-PTH fragments, resulting in a high C-PTH/(1–84)hPTH ratio in the circulation (8, 9, 10). PTH degradation occurs within the parathyroid glands before secretion (11), although peripheral metabolism is also observed after secretion (12). Secretion of (1–84)hPTH is controlled by a Ca2+-dependent degradation process (13, 14, 15), which involves destruction of secretory granule contents in lysosomes and possibly also in secretory granules as well (16, 17). Although in vivo (8, 18) and in vitro (7, 11, 19, 20) studies have demonstrated that extracellular Ca2+ induces PTH cleavage to generate C-terminal fragments such as (34–84)hPTH, N-terminal truncation to generate large carboxyl-terminal fragments or non-(1–84)hPTH has not been previously reported.

Both the (1–84)hPTH molecule and N-terminally truncated fragments such as (7–84)hPTH can be measured by commercially available two-site intact PTH (I-PTH) assays (21, 22). Recently, the bio-intact PTH (Bio-PTH) assay, a new two-site immunochemiluminometric assay, which recognizes only (1–84)hPTH has been developed (23, 24). Interestingly, the (1–84)PTH/I-PTH ratio is negatively correlated with serum Ca2+ level in patients on maintenance hemodialysis (25), suggesting that serum Ca2+ concentration may regulate the degradation of (1–84)hPTH by N-terminal truncation in parathyroid cells or in the peripheral metabolism. Although intracellular degradation of PTH in response to increases in serum Ca2+ levels could be one mechanism to suppress (1–84)hPTH secretion, the importance of intracellular N-terminal truncation has not been elucidated.

In this study, we attempted to identify the actual N-terminally truncated PTH fragments by HPLC and to clarify the regulation of intracellular N-terminal truncation by extracellular Ca2+ in vitro.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

This study included four patients with primary hyperparathyroidism (PHPT) and three with uremia-associated secondary hyperparathyroidism (SHPT) who underwent therapeutic parathyroidectomy at Osaka City University Hospital or Shirasagi Hospital between 2003 and 2004. The PHPT patients were all diagnosed as having parathyroid adenomas. All participants met the criteria for therapeutic parathyroidectomy for PHPT (26) or SHPT (27). All subjects provided written informed consent before participation in this study, which was approved by the institutional ethics committees of Osaka City University Graduate School of Medicine and Shirasagi Hospital and was conducted in accordance with the principles of the Declaration of Helsinki.

Preparation of human parathyroid cell culture

Isolation of human parathyroid cells for primary monolayer cell cultures was performed as described previously (28), with some modifications (29). Parathyroid glands excised from PHPT patients or the largest removed glands from SHPT patients were used for the cultures. In both cases, tiny parts of the glands were used to diagnose the pathologies. Briefly, dispersed parathyroid cells were cultured overnight in Ham-F10 medium containing 4% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. The concentration of Ca2+ in the culture medium was 0.5 mM. After overnight culture to allow the cells to adhere to the plates, the cells were treated with media containing 0.5, 1.0, 2.0, or 3.0 mM Ca2+ for 6 h without fetal bovine serum and then washed twice with PBS to remove the previously secreted PTH. After a 1-h incubation with fresh medium, the media from all treatments were collected and stored at –20 C until assays were performed.

Incubation of exogenously added (1–84)PTH with cultured parathyroid cells

Full-length hPTH, (1–84)hPTH (Peptide Institute, Inc., Osaka, Japan), was incubated with cultured parathyroid cells for 1 h. The concentration of exogenous (1–84)hPTH was 50,000 pg/ml in the medium, which is approximately 10–20 times higher than that secreted by the cultured cell in 1 h.

HPLC analysis of the PTH fraction

The media were applied to a C18-SepPack column equilibrated immediately before use with 0.1% trifluoroacetic acid (TFA) buffer followed by elution with 80% acetonitrile with 0.1% TFA buffer. The eluate was dried and dissolved in PBS with 0.7% BSA for HPLC analysis. Separations were performed a using µBondapac C18 column (Waters Chromatographic Division, Milford, MA). Elution was performed by a gradient system with two solvents (solvent A, 0.1% TFA/HPLC water; solvent B, 0.1% TFA/CH3CN). Eluate was collected in 1-min fractions. Full-length (1–84)hPTH (Japan Peptide Research Institute) and (7–84)hPTH (Bachem AG, Bubendorf, Switzerland) were used as standards. The samples were dried, dissolved in PBS with 0.1% BSA, and stored at –20 C until assays were performed.

Measurement of PTH concentration

The media and the HPLC eluate were tested by two different methods of PTH measurement, using the I-PTH and the Bio-PTH assays. The I-PTH assay was performed using the Allegro Intact PTH Kit (Nichol’s Institute, San Juan Capistrano, CA). The Bio-PTH assay is a newly developed two-site immunochemiluminometric assay that measures (1–84)hPTH exclusively, in contrast to the I-PTH assay (24). Bio-PTH is a sandwich assay using an acridinium ester-labeled goat anti-PTH antibody and a biotinylated capture antibody that complexes with (1–84)hPTH alone.

Statistical analysis

All results are means ± SD of triplicate determinations. Statistical significance was determined by Student’s t test or Dunnett’s test using commercially available software for Windows (StatView V; SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
PTH fragmentation in cultured parathyroid cells in vitro

To identify the PTH fragments that were detected by the Bio-PTH and I-PTH assays, samples of parathyroid adenoma cell culture media were separated by HPLC. First, we analyzed the immunoreactive peaks obtained from (1–84)hPTH and from (7–84)hPTH using both Bio-PTH and I-PTH assays. An immunoreactive peak was detected in the same elution position (fraction 20) using both assays and represented the full-length (1–84)hPTH (Fig. 1AGo). Although an earlier immunoreactive peak (fraction 17) caused by (7–84)hPTH was detected using the I-PTH assay, no corresponding peak was detected using the Bio-PTH assay (Fig. 1BGo).



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  		FIG. 1. HPLC profiles of immunoreactive PTH in full-length (1–84)hPTH and truncated (7–84)hPTH. Immunoreactive peaks were obtained for (1–84)hPTH (A) and (7–84)hPTH (B) using the Bio-PTH and I-PTH assays. An immunoreactive peak was detected by both assays in the same elution position (fraction 20, white arrow) upon elution of a (1–84)hPTH standard (A). An earlier immunoreactive peak (fraction 17, black arrow) was detected using a (7–84)hPTH standard in the I-PTH assay, but no such peak was detected using the Bio-PTH assay (B).

 
Next, we analyzed samples of media from parathyroid adenoma cell cultures that had been preincubated for 6 h with various Ca2+ concentrations and then incubated for 1 h in the fresh media with the same Ca2+ concentrations. Degraded non-(1–84)hPTH fragments were not detected in media with 0.5 mM Ca2+ (Fig. 2AGo). The immunoreactive peak of non-(1–84)hPTH was observed at 1.0 mM Ca2+ (Fig. 2BGo) and 2 mM Ca2+, in which the non-(1–84)PTH peak was greater than that of (1–84)hPTH (Fig. 2CGo). Only the immunoreactive (1–84)hPTH peak was observed in media assayed using the Bio-PTH assay. No peaks other than (1–84)hPTH and non-(1–84)hPTH were observed in media using the I-PTH assay.



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  		FIG. 2. HPLC profiles of immunoreactive PTH present in culture medium from parathyroid adenomas. Parathyroid adenoma cells were pretreated in media with various concentrations of Ca2+ (0.5, 1.0, or 2.0 mM) for 6 h, followed by a 1-h incubation with identical media to measure PTH secretion profiles. The elution positions of (1–84)hPTH and (7–84)hPTH are indicated by white and black arrows, respectively. The non-(1–84)hPTH fragment was not detected in media with 0.5 mM Ca2+ (A). The immunoreactive peak of non-(1–84)hPTH was observed at 1.0 mM Ca2+ (B), and at 2.0 mM Ca2+, the peak of non-(1–84)hPTH was higher than that of (1–84)hPTH (C). Only the (1–84)hPTH immunoreactive peak was observed with the Bio-PTH assay. No peaks other than these two peaks were detected by the I-PTH assay.

 
Effect of extracellular Ca2+ on PTH secretion and degradation in cultured parathyroid cells

To assess the intracellular degradation of PTH associated with various concentrations of extracellular Ca2+, cultured cells from parathyroid adenomas or uremia-associated SHPTs were analyzed. With both Bio-PTH and I-PTH, detectable PTH secretion decreased in a concentration-dependent manner between Ca2+ concentrations of 0.5 and 3.0 mM (Fig. 3Go, A and B). In parathyroid adenomas, PTH secretion was significantly suppressed with both 2.0 and 3.0 mM Ca2+ compared with 0.5 mM Ca2+, as measured by both Bio-PTH and I-PTH assays (Fig. 3AGo). The ratios of inhibitory effects on PTH secretion at 2.0 or 3.0 mM Ca2+ to that at 0.5 mM Ca2+ were significantly higher with the Bio-PTH than with the I-PTH assay.



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  		FIG. 3. Concentration-dependent inhibitory effects of extracellular Ca2+ on PTH secretion in cultured parathyroid cells. Parathyroid cells prepared from parathyroid adenomas (A) or uremia-associated SHPTs (B) were pretreated in media with various concentrations of Ca2+ (0.5, 1.0, or 2.0 mM) for 6 h, followed by a 1-h incubation with identical media to measure PTH secretion profiles. Both the Bio-PTH and I-PTH assays measured a concentration-dependent decrease in PTH in Ca2+ concentrations between 0.5 and 3.0 mM (A and B). The inhibitory effects of Ca2+ on PTH secretion were significantly stronger as measured by the Bio-PTH assay than as measured by the I-PTH assay for both types of parathyroid cells. Values are means ± SD of three different cultures in triplicate. *, P < 0.01; **, P < 0.001 vs. Ca2+ 0.5 mM by Dunnett’s test. ¶, P < 0.01 between I-PTH and Bio-PTH at each Ca2+ concentration by Student’s t test.

 
In uremia-associated hyperparathyroidism, inhibitory effects on PTH secretion were observed with 2.0 mM Ca2+ or higher using the I-PTH assay but were observed at lower Ca2+ concentrations (1.0 mM Ca2+ or higher) using the Bio-PTH assay (Fig. 3BGo). The inhibitory effects on PTH secretion at 1.0, 2.0, or 3.0 mM Ca2+ compared with that at 0.5 mM Ca2+ were significantly higher when measured by the Bio-PTH assay than when measured by the I-PTH assay.

To investigate the effect of extracellular Ca2+ on PTH degradation, the Bio-PTH/I-PTH ratio was calculated as an index of PTH degradation for each Ca2+ concentration (Fig. 4Go). Bio-PTH/I-PTH ratios were suppressed in a Ca2+ concentration-dependent manner in both parathyroid adenomas and uremia-associated parathyroid tumors. The ratios were 71.8 and 70.0% at 0.5 mM Ca2+, 56.7 and 48.8% at 1.0 mM Ca2+, 46.2 and 36.9% at 2.0 mM Ca2+, and 36.9 and 36.8% at 3.0 mM Ca2+ for parathyroid adenomas and uremia-associated parathyroid tumors, respectively. Bio-PTH/I-PTH ratios did not differ significantly between cell cultures obtained from these two parathyroid diseases for any Ca2+ concentration tested.



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  		FIG. 4. Suppressive effects of extracellular Ca2+ on Bio-PTH/I-PTH ratios in cultured parathyroid cells. To investigate the effect of extracellular Ca2+ on PTH degradation, the Bio-PTH/I-PTH ratio was calculated as an index of PTH degradation. Bio-PTH/I-PTH ratios were reduced in a Ca2+ concentration-dependent manner in cells from both parathyroid adenomas and uremia-associated parathyroid tumors. The differences in Bio-PTH/I-PTH ratios between these two parathyroid diseases were not significant at any of the Ca2+ concentrations tested. Values are means ± SD of three different cultures in triplicate. *, P < 0.01; **, P < 0.001 vs. Ca2+ 0.5 mM by Dunnett’s test.

 
Effect of extracellular Ca2+ on degradation of PTH by cultured parathyroid cells

To determine whether extracellular Ca2+-regulated PTH degradation occurred primarily through an intracellular or an extracellular mechanism, exogenous (1–84)hPTH at a final concentration of 50,000 pg/ml was incubated with the parathyroid cells for 1 h. No significant difference of Bio-PTH/I-PTH ratio was observed in culture media from parathyroid cells incubated with Ca2+ concentrations between 0.5 and 2.0 mM (Fig. 5Go).



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  		FIG. 5. Effect of extracellular Ca2+ on degradation of (1–84)hPTH by cultured parathyroid cells. To determine whether the site of (1–84)hPTH degradation was intracellular or extracellular, exogenous (1–84)hPTH (50,000 pg/ml) was added to the medium of the parathyroid cell cultures for 1 h. No significant difference in Bio- PTH/I-PTH ratio was observed between 0.5 and 2.0 mM Ca2+. Values are means ± SD of four different wells of cultured cells from the same parathyroid.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This study was designed to evaluate the effect of extracellular Ca2+ concentration on the production of N-terminal truncations of PTH. The I-PTH assay detected both the full-length (1–84)hPTH and the N-terminally truncated, large carboxyl-terminal fragments, non-(1–84)hPTH, which are secreted from parathyroid cells. Increases in extracellular Ca2+ accelerated production of N-terminal truncations of (1–84)hPTH, suggesting that N-terminal truncation may have a physiological role in the suppression of (1–84)hPTH secretion. When excess exogenous (1–84)hPTH was incubated with parathyroid gland cells at various Ca2+ concentrations, changes in extracellular Ca2+ did not alter the Bio-PTH/I-PTH ratio in the culture medium, suggesting that N-terminal truncation of (1–84)hPTH occurred primarily by an intracellular mechanism.

Our HPLC study clearly indicated that N-terminal truncation occurred in parathyroid cells, with the non-(1–84)hPTH fragments detectable by I-PTH assay. In addition, this degradation was enhanced by incubating the cells in high Ca2+, suggesting that extracellular Ca2+ directly increases PTH degradation to suppress levels of bioactive (1–84)hPTH. Although previous studies in humans (10) and dogs (30) indicated that extracellular Ca2+ accelerated PTH degradation, resulting in increased C-terminal fragment/I-PTH ratio, the PTH cleavage site in these studies was in the midregion of the PTH molecule, which produces a cleavage product undetectable by the I-PTH assay. HPLC studies also revealed a shift of the immunoreactive PTH peak with changes in the extracellular Ca2+ level (9, 11, 14, 18, 20), and although these studies suggested the existence of midregion cleavages in PTH molecules, N-terminal truncations were not examined.

In this study, we investigated the N-terminal truncation of (1–84)hPTH, and not midregion cleavages, and determined the importance of stimulation of N-terminal truncation induced by extracellular Ca2+. Comparison of PTH concentrations measured by the Bio-PTH and I-PTH revealed that elevation of extracellular Ca2+ suppressed PTH secretion, although the level of suppression differed depending on the assay used to measure PTH. Similar results were obtained for both parathyroid adenomas and uremia-associated SHPT. The Bio-PTH assay was superior to the I-PTH assay because the Bio-PTH detected only full-length (1–84)hPTH. N-terminally truncated non-(1–84)PTH interfered with the I-PTH assay and decreased the reduction of PTH measured in the assay. In fact, the Bio-PTH/I-PTH ratio decreased in an extracellular Ca2+ concentration-dependent manner. These findings are also consistent with the N-terminal truncations observed in the HPLC study. Peripheral degradation also plays a role in N-terminal truncation of (1–84)hPTH (12). A negative correlation between serum Bio-PTH/I-PTH ratio and serum Ca2+ level has been observed in maintenance hemodialysis patients (25). N-terminal truncation of (1–84)hPTH in parathyroid cells or in peripheral tissues might account for this negative correlation in hemodialysis patients. These mechanisms of N-terminal truncation are physiologically relevant to Ca2+ homeostasis, because non-(1–84)hPTH fragments such as (7–84)hPTH inhibit the action of (1–84)hPTH in bone (31). High serum Ca2+ levels could accelerate degradation to reduce (1–84)hPTH and raise non-(1–84)hPTH concentrations such as the (7–84)hPTH fragment, resulting in attenuation of PTH action caused by reduction of (1–84)hPTH levels and skeletal resistance to (7–84)hPTH.

Calcimimetics such as cinacalcet HCl, an allosteric modulator of the CaR, have met with great success as novel drugs for the treatment of SHPT (32, 33) and PHPT (34, 35) in clinical studies, despite the fact that reduced expression of CaR in parathyroid glands has been observed in PHPT and SHPT (36, 37). The calcimimetic compound NPS R-568 rapidly inhibited PTH secretion in dispersed bovine parathyroid cells in vitro (38). CaR activation might be involved in production of N-terminal truncations, suggesting that the truncation could partly contribute to the inhibitory effect of calcimimetics on PTH secretion. Although extracellular Ca2+ or calcimimetics induce a prompt suppression of PTH secretion from parathyroid gland, the physiological significance of the decrease in full-length (1–84)hPTH is yet to be determined.

In conclusion, this study provides new findings on accelerated production of N-terminal truncations of PTH in diseased parathyroid cells resulting from increases in extracellular Ca2+.


   Acknowledgments
 
We thank Kyohya Akiko and Kinya Fujishiro of Kyowa Medics, Inc., Tokyo, Japan, for their excellent technical support.


   Footnotes
 
First Published Online July 26, 2005

Abbreviations: Bio-PTH, Bio-intact PTH; CaR, calcium-sensing receptor; C-PTH, C-terminal PTH; hPTH, human PTH; I-PTH, intact PTH; PHPT, primary hyperparathyroidism; SHPT, secondary hyperparathyroidism; TFA, trifluoroacetic acid.

Received February 3, 2005.

Accepted July 15, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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