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Keywords:

  • parathyroid;
  • parathyroid hormone;
  • calcium;
  • calcium-sensing receptor;
  • three-dimensional collagen

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Primary cultures of bovine parathyroid cells rapidly lose calcium responsiveness. Here, we show that bovine parathyroid cells grown in collagen coalesce into an organoid (“pseudogland”) with stable calcium responsiveness. These findings also illustrate the importance of 3-D cellular architecture in parathyroid gland function.

Introduction: The ability of extracellular calcium to suppress parathyroid hormone (PTH) secretion is quickly lost in primary monolayer cultures of bovine parathyroid cells. This has been attributed to a decrease in the expression of the cell surface calcium-sensing receptor (CaR), but other factors, including normal cell-to-cell interaction, may be critical. Here we describe a novel system for culturing bovine parathyroid cells that promotes re-formation of a three-dimensional (3-D) cellular architecture and re-establishment of calcium responsiveness.

Materials and Methods: Dispersed bovine parathyroid cells were cultured as monolayers or were mixed with type I collagen and placed in culture plates. CaR mRNA and the calcium regulation of PTH secretion were measured over a period of several weeks in parathyroid cells cultured both in collagen matrix and as monolayers. Calcium regulation of PTH mRNA was also investigated.

Results and Conclusions: Within 1–2 weeks in collagen culture, parathyroid cells coalesced into a small mass approximately 1–2 mm in size (referred to as a pseudogland). Suppression of PTH secretion by high calcium was blunted at 1 day in collagen, but returned within 1 week, and was retained through 3 weeks; the calcium set point (1.05 ± 0.04 mM) was similar to that reported for freshly dispersed cells. PTH mRNA was also suppressed by increasing extracellular calcium. CaR mRNA expression was decreased at 1 day in collagen and increased with time in culture, although never reaching the level found in dispersed cells. In bovine parathyroid cells cultured as monolayers, however, suppression of PTH by calcium was observed only at day 1 in culture. CaR mRNA content fell by 70% at day 1 but remained stable thereafter. Thus, a total loss of calcium responsiveness in monolayers was observed despite significant residual expression of CaR, suggesting that loss of the calcium response cannot be attributed solely to decreased CaR. In summary, the pseudogland model illustrates the importance of the 3-D cellular architecture in parathyroid gland function and provides a useful model in which to investigate calcium-mediated control of parathyroid gland functions, especially those requiring extended treatment.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

CHIEF CELLS OF the parathyroid gland express a surface calcium-sensing receptor (CaR) that detects small changes in serum calcium and controls the rate of parathyroid hormone (PTH) secretion.(1, 2) Our current understanding of the acute calcium regulation of PTH and parathyroid signaling pathways emanating from the CaR has been obtained primarily through the use of freshly isolated parathyroid cells. Because there are no parathyroid cell lines that secrete PTH in a calcium-dependent manner, the chronic effects of calcium on parathyroid cell function have been more difficult to study. Bovine parathyroid cells quickly lose their response to calcium when placed in primary monolayer culture.(3, 4) This has been attributed to a dramatic decrease in the expression of the CaR that occurs on collagenase dispersal and subsequent culturing of the bovine parathyroid cells in monolayers.

Culturing cells in a manner that preserves or provides a more natural three-dimensional (3-D) environment has been shown to maintain certain cellular functions in parathyroid cells. Nearly 20 years ago, Ridgeway et al.(5) found that bovine parathyroid cells cultured as multicellular aggregates (termed organoids) maintained their tissue-like morphology and differentiated function, including their response to extracellular calcium, for at least 2 weeks in vitro. This culturing system, which required the use of roller bottles, was relatively simple to initiate and maintain, and provided the means by which the modulation of parathyroid function could be studied over an extended period of time in culture. In other studies, multicellular aggregates of human parathyroid cells grown as monolayers, obtained from tissue of patients with secondary hyperparathyroidism, responded to calcium for up to 5 months,(6) but these cells are known to harbor mutations that may influence the end points to be studied.(7) A direct stimulatory effect of phosphate on PTH secretion in vitro has been demonstrated in intact rat glands and bovine tissue slices but not in dispersed parathyroid cells or monolayer cultures.(8–10) Taken together, these findings indicate that a higher level of tissue structure in culture can retain at least some of the parathyroid cellular functions.

Culturing cells in a 3-D layer of collagen has been found to promote reorganization of cells into structures reminiscent of their native state and can induce differentiated cell function in a variety of cell types. When grown in collagen, the colon carcinoma cell line SW1222 formed glandular structures of cells organized around a central lumen.(11) These cells exhibited epithelial polarity and mucin production similar to that seen in normal intestinal glands. The well-differentiated human breast carcinoma T47D cell line organized in a manner suggestive of glandular epithelium when cultured in collagen gels.(12) Non-tumor cells have also been used in similar culture systems. When placed in a 3-D collagen gel matrix, guinea pig enteric ganglia maintain the organization of neurons and glial cells within myenteric ganglia, as well as the ability to organize into an orderly network of ganglia and interconnecting strands.(13) Adult human tracheal cells, when cultured in a collagen gel matrix, form functional tracheal gland-like tubules.(14) In addition, in a 3-D collagen culture, thyrocytes form stable follicles with physiological polarity, whereas in monolayer and floating culture systems they do not.(15) Therefore, in certain cell types, the 3-D collagen culture system provides the cell-cell and/or cell-extracellular matrix interactions that may be necessary for retention of differentiated cellular functions.

In an attempt to develop more differentiated parathyroid cells in culture, one in which parathyroid cells would retain the ability to respond to extracellular calcium, bovine parathyroid cells were cultured in a 3-D collagen gel matrix.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Preparation of dispersed bovine parathyroid cells

Dispersed bovine parathyroid cells were prepared as previously described.(16) Briefly, bovine parathyroid glands were trimmed of extraneous fatty tissue, sliced to 0.5 mm thickness with a tissue slicer (Stadie Riggs; Thomas Scientific), and placed in a mixture of DME:Ham's F-12 medium (50:50) containing 0.5 mM calcium and collagenase (3000 U/ml of collagenase XI-S; Sigma). The suspension (10 ml media/g of tissue) was agitated in a shaking water bath at 37°C for 90 minutes. Periodically passing the mixture through the tip of a 10-ml pipette assisted in the disaggregation. The digested tissue was filtered through gauze, resuspended, and washed three times with serum-free culture medium containing DME:Ham's F-12 (50:50; 1 mM calcium, 0.7 mM magnesium), 15 mM HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, 5 μg/ml holo-transferrin, 2 mM glutamine, and 1% nonessential amino acids. With the exception of changes in calcium concentration or the addition of serum, as specified in the text, this is the culture medium used for all experiments. For measurement of PTH secretion by freshly dispersed parathyroid cells, the cells were incubated in serum-free culture medium (0.2 × 106 cells/0.5 ml) for indicated amounts of time at 37°C. The cells were centrifuged, and the supernatant was frozen for PTH analysis.

3-D collagen culture

On ice, the dispersed parathyroid cells were added to a 1:1 mixture of rat-tail type I collagen (BD Biosciences) and 2× Krebs-Henseleit buffer and placed in 24-well culture plates with a total volume of 0.25 ml/well. Each well contained 0.2 × 106 cells, and the final collagen concentration was 1.45 mg/ml. The collagen/cell mixtures were allowed to solidify at 37°C in a 5% CO2 incubator, after which 0.25 ml/well of serum-free medium was carefully layered on top. The collagen/cell mixtures were left undisturbed until coalescence was complete (1–2 weeks). Medium was changed after the cells coalesced and weekly thereafter. Pseudoglands used in PTH secretion studies were between 1 and 4 weeks old.

Parathyroid monolayer cultures

Dispersed cells were plated in 12-well culture plates at 0.2 × 106 /well in the above medium containing 4% newborn calf serum. After 24 h, the medium was replaced with serum-free medium. Medium was changed every 2–3 days of culture.

PTH secretion

To examine PTH secretion, media from monolayers or collagen cultures were collected on ice and centrifuged, and supernatants were stored at −20°C until PTH analysis was performed. Immunoreactive PTH was assayed using antiserum CH9, which was raised against bovine PTH and recognizes intact PTH, midregion, and COOH-terminal fragments, as described previously.(17) DNA was measured by the ethidium bromide method of Le Pecq and Paoletti.(18)

Measurement of CaR mRNA and PTH mRNA by ribonuclease protection assay

CaR mRNA in bovine parathyroid cells was analyzed using our previously described ribonuclease protection assay.(3) Briefly, RNA from dispersed parathyroid cells, monolayers, or pseudoglands was mixed with the32P-labeled riboprobes for CaR and β-actin. After hybridization at 45°C for 16 h, the samples were digested with ribonuclease T-1 and then with proteinase K. The protected RNA fragments were extracted with phenol-chloroform, precipitated with ethanol, and resolved on a 5% polyacrylamide gel. The gel was dried, and the bands were quantified using a phosphorimager (model 445; Molecular Dynamics). The template for bovine PTH was made by reverse transcriptase-polymerase chain reaction (RT-PCR) of bovine parathyroid gland RNA and consisted of 297 bp corresponding to bp 253–449 of the published sequence for bovine PTH cDNA (accession no. M25082).(19) The assay was performed as described above for CaR mRNA. CaR mRNA and PTH mRNA were normalized to β-actin mRNA.

Immunostaining

Rat parathyroid glands, bovine parathyroid tissue, and bovine pseudoglands were fixed in 10% buffered formalin for 24 h and placed in 70% EtOH. Dispersed bovine parathyroid cells (∼1 × 106 cells) were centrifuged in a conical 15-ml tube, and the resulting pellet was placed intact into 10% buffered formalin for 24 h and then switched to 70% EtOH. Hematoxylin-eosin (H&E) staining was performed on formalin-fixed, paraffin-embedded sections of the bovine parathyroid tissue, bovine parathyroid dispersed cells, bovine pseudoglands, and an intact rat parathyroid gland.

Formalin-fixed, paraffin-embedded sections of the bovine pseudoglands were analyzed for CaR by immunostaining using a commercial kit (Histostain-Plus kit; Zymed) and a 1° antibody directed against the extracellular domain of the CaR as previously described.(20) Briefly, sections were deparaffinized and rehydrated, and endogenous peroxidase was quenched using 0.6% hydrogen peroxide in methanol. Tissue was blocked with 10% nonimmune goat serum and incubated overnight at 4°C with 1° antibody or preimmune IgG. Biotinylated 2° antibody was applied, followed by streptavidin-horseradish peroxidase conjugate. The reaction was visualized with 3-amino, 9-ethyl-carbazole (AEC) substrate-chromagen.

Statistics

Statistical significance of the differences between means was evaluated by Student's t-test, (Instat; Graph Pad Software, San Diego, CA, USA), except for the set-point experiments in which one-way analysis of variance was used.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Coalescence of bovine parathyroid cells cultured in collagen

Bovine parathyroid cells were mixed with rat-tail type I collagen and placed at 37°C to allow the matrix to solidify. Within the first few days in culture, the collagen gel became more liquid in appearance and began to detach from the culture well. This detachment began at the sides of the culture well and progressed toward the center of the well. The fully detached gel then floated freely in the overlay media, and the entire mass began to shrink in size. This coalescence of the cells continued, and over a period of 1–2 weeks, a tight ball of cells approximately 1-2 mm in diameter was formed. We refer to this mass of cells as a pseudogland. This process of coalescence is shown in Fig. 1. Figure 1A shows the solidified collagen gel/parathyroid cell mixture at day 1, partial coalescence at 5 days, and a fully coalesced pseudogland after 2 weeks in culture. Figure 1B shows H&E staining of paraffin sections of the coalescing parathyroid cells at 2, 5, and 14 days in collagen culture. The infrastructure of a pseudogland is shown in Fig. 2, which compares H&E-stained sections of intact bovine parathyroid tissue, dispersed bovine parathyroid cells, a bovine pseudogland, and a rat parathyroid gland. The architecture of the pseudogland is remarkably similar to that of the intact bovine parathyroid gland and the intact rat parathyroid gland, suggesting that formation of the pseudogland involves reconstruction of extracellular matrix in addition to compaction of the cells during coalescence. This is in obvious contrast to the dispersed bovine parathyroid cells, which lack extracellular matrix and cell-cell adhesion.

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Figure FIG. 1.. Formation of a bovine parathyroid pseudogland. (A) Intact collagen gel/cell mixture shortly after solidification and the subsequent coalescence of the cells at 5 and 14 days in culture; magnification, ×100. (B) H&E stain of paraffin sections of the cells at 2, 5, and 14 days of coalescence; magnification, ×100.

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Figure FIG. 2.. Comparison of sections of a bovine parathyroid (PT) gland, dispersed bovine PT cells, a fully formed bovine pseudogland, and a rat PT gland. H&E stain, magnification, 400×.

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To ensure proper formation of the bovine parathyroid pseudoglands, it is important that they not be disturbed during the coalescence. Excessive handling (such as swirling the contents of the well) early in the process of coalescence seemed to break the newly forming bonds between the cells and inhibited formation of the pseudogland. Therefore, once plated, the collagen/cell mixtures were left undisturbed until coalescence was nearly complete. Once the pseudoglands were formed, they were easily transferred to a fresh well or test tube using a wide bore pipette.

Two other factors seemed to influence formation of the pseudoglands. First, medium containing serum inhibited coalescence of the cells (data not shown). When 4% newborn calf serum was added to the overlay medium (layered over the collagen/cell mixture once it solidified), the process of coalescence was completely inhibited. The reason for this is unknown. The other factor that influenced coalescence was the type of collagenase used. Samples of collagenase from several companies were tested; the quality of pseudogland formation was variable among all types tested and sometimes varied between lot numbers of the same type. The collagenase referenced in the Materials and Methods section reflects the most recently used collagenase that has given very reproducible, reliable coalescence.

Calcium responsiveness of PTH secretion by pseudoglands

Calcium-dependent suppression of PTH secretion was examined in the parathyroid pseudoglands. In a representative experiment, 12-day-old pseudoglands were washed three times with fresh media and placed for 4 h in media containing ionized calcium (Ca2+) concentrations ranging from 0.4 to 3.7 mM. The media were collected for analysis of immunoreactive PTH; data are reported as mean ± SD. As shown in Fig. 3, maximal PTH secretion at 0.4 mM calcium was 43.5 ± 7.3 ng/ml and minimal secretion at 3.7 mM calcium was 19.1 ± 1.9 ng/ml. The set point, the calcium concentration at which the rate of PTH was 50% of the maximal-minimal secretion (31.3 ng/ml), was 1.00 mM calcium. This was reproduced in two other experiments performed under similar conditions. The average set point ± SD for the three experiments was 1.05 ± 0.04. PTH secretion by pseudoglands incubated in medium containing calcium of 1 mM was found to be linear during a 4-h period (r = 0.991, p < 0.01; data not shown).

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Figure FIG. 3.. Set point for PTH secretion in pseudoglands. Twelve-day-old pseudoglands were incubated for 4 h in varying amounts of ionized calcium (n = 6 pseudoglands per calcium concentration). The set point (50% of maximal-minimal PTH) was 1.00 mM calcium. Data reported as mean ± SD; p < 0.01 compared with the 0.4 mM calcium concentration.

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PTH secretion in pseudoglands and monolayers

The PTH response to calcium was measured at 1 week in culture for both pseudoglands and bovine parathyroid cell monolayers. Cells from each culture system were washed three times with medium and replenished with low (0.5 mM) or high (3 mM) calcium for 1 h. PTH was normalized with DNA, and results are shown in Fig. 4. A 43% suppression of PTH was seen in the pseudoglands (p < 0.01). There was no response to calcium in the monolayer cultures. This figure is representative of at least 20 separate experiments, with PTH analysis performed between 1 and 3 weeks in culture.

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Figure FIG. 4.. PTH secretion in bovine pseudoglands and monolayers. After 1 week in culture, bovine pseudoglands and monolayers were challenged with media containing low (0.5 mM) and high (3.0 mM) concentrations of calcium; n = 6 per condition. A significant suppression of PTH by high calcium was seen in the pseudoglands (*p < 0.01). No response to calcium was observed in monolayer cultures. Data reported as mean ± SD.

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Effect of calcium on PTH mRNA in pseudoglands

The effects of calcium on the expression of PTH mRNA in the bovine parathyroid pseudoglands are shown in Fig. 5. Ten-day-old pseudoglands (1–2 mm in size) were incubated for 72 h with low (0.5 mM), normal (1.0 mM), and high (3.0 mM) calcium. Compared with normal calcium, a significant increase in PTH mRNA was seen at low calcium (25%, p < 0.001), and a significant decrease was seen at high calcium (76%, p < 0.0001). Significant changes in PTH secretion corresponded to changes in the PTH mRNA (data not shown). Similar changes in PTH mRNA were obtained in a separate experiment using a 48-h incubation with low, normal, and high calcium.

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Figure FIG. 5.. Effect of calcium on PTH mRNA in pseudoglands. PTH mRNA (corrected with actin mRNA) was measured in bovine parathyroid pseudoglands by ribonuclease protection assay. Pseudoglands were incubated for 72 h with low (0.5 mM), normal (1.0 mM), or high (3.0 mM) calcium. Compared with normal calcium, a significant increase in PTH mRNA was seen at low calcium (25%, p < 0.001), and a significant decrease was seen at high calcium (76%, p < 0.0001). Data reported as mean ± SD; n = 5 points per condition; 3 pseudoglands per point.

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Time course of DNA content in pseudoglands and monolayers

DNA content was measured in freshly dispersed bovine parathyroid cells, and weekly for 4 weeks in pseudoglands and monolayers. The DNA content of the pseudoglands at 1 week in culture (3.06 ± 0.61 μg/pseudogland) was not different from the DNA content of the dispersed cells (3.17 ± 0.33 μg/200,000 cells) and did not change significantly over 4 weeks (3.29 ± 0.12 μg/pseudogland at 4 weeks). However, the DNA content in monolayers increased 4-fold over the same period of time (3.6 ± 0.21 at 1 week to 11.4 ± 0.59 μg/well at 4 weeks; p < 0.0001). The finding that DNA does not increase with time in culture in the pseudoglands would indicate that they more closely resemble normal parathyroid tissue in vivo (which rarely proliferates) than the more rapidly growing parathyroid cells of monolayers.

Time course of PTH secretion in pseudoglands and monolayers

The PTH response to calcium was measured in freshly dispersed bovine parathyroid cells and at progressive times in culture when cells were placed in collagen or plated as monolayers. At each time point, cells were washed three times with medium and replenished with low or high calcium (0.2 and 3 mM, respectively) for 1 h. After media were collected for PTH secretion, the pseudoglands were harvested for DNA content. It should be noted that the intact collagen gel interferes in the DNA assay, thus preventing measurement of DNA content of the cells at 1 day in collagen culture. Therefore, to correct PTH secretion with DNA at this time point, the DNA content of the dispersed cells was used. (The same number of cells was used for all time points. Because there was no difference in the DNA content of the dispersed cells and the DNA content of the pseudoglands at 1, 2, and 3 weeks in culture, there was no reason to assume that the DNA content of the cells in culture at 1 day in culture would be different.) PTH was corrected for DNA, and results are reported as percent suppression by 3 mM calcium. As shown in Fig. 6, there was a 54% suppression of PTH by high calcium in the dispersed cells. In cells cultured in collagen, there was no suppression of PTH secretion at 1 day, at which time the cells are still embedded in the solid collagen matrix. However, calcium responsiveness returned by 1 week in culture, when collagen matrix breakdown and coalescence of the cells into pseudoglands was well underway, if not nearly complete. The response to calcium persisted in the pseudoglands through 3 weeks in culture. In parathyroid cells plated as monolayers, there was a response to 3 mM calcium at 1 day in culture, but not thereafter. Thus, culturing bovine parathyroid cells in a collagen matrix promotes expression of the calcium response once the cells reorganize into a structure that is reminiscent of intact parathyroid tissue. This is in contrast to cells cultured as monolayers, which quickly lose the response to calcium.

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Figure FIG. 6.. Time course of PTH secretion in pseudoglands and monolayers. The PTH response to calcium was measured in freshly dispersed bovine parathyroid cells and at progressive time points in collagen and monolayer culture. At each time point, cells were challenged with 0.2 or 3 mM calcium for 1 h. PTH secretion was corrected with DNA and reported as percent suppression. There was significant suppression of PTH secretion by high calcium in dispersed cells but not at 1 day in collagen culture. However, the response to calcium returned by 1, 2, and 3 weeks in culture in the pseudoglands. In the monolayers, there was a response to calcium at 1 day in culture, but not thereafter. These data are representative of four experiments. In one experiment, the calcium response persisted through 6 weeks in culture. (*p < 0.05, **p < 0.01; n = 6 per condition).

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Time course of CaR mRNA expression in parathyroid pseudoglands and monolayers

Because the bovine parathyroid pseudoglands retain a response to calcium, it was hypothesized that they would express high levels of CaR. Therefore, CaR mRNA was measured in freshly dispersed bovine parathyroid cells and at progressive times in culture when cells were placed in collagen or plated as monolayers. Results are shown in Fig. 7. Although initially decreased in the collagen culture at 1 day, a modest, gradual increase in CaR mRNA with time in culture was observed; at 3 weeks in culture, the pseudogland CaR mRNA increased to 60% of dispersed cell values and remained near this level through 4 weeks in culture. The CaR mRNA in monolayers also decreased by 1 day in culture but did not increase with time in culture; at 3 weeks, the monolayer CaR mRNA was approximately 30% of dispersed cell values, and only ∼50% of that expressed by the pseudoglands. Similar results were seen in a replicate experiment. It is interesting that, although a modest level of CaR remained at 4 weeks in the pseudoglands, calcium was not able to suppress PTH at this time point (see Fig. 6). In addition, although there is a dramatic decrease in CaR expression in the monolayers at 1 day in culture, the calcium response is still intact; however, the response is lost quickly thereafter (as shown in Fig. 6), with relatively little further change in CaR expression. This may indicate that loss of the calcium response may be caused by factors other than just loss of CaR expression, such as changes in downstream signaling.

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Figure FIG. 7.. Time course of CaR mRNA expression in pseudoglands and monolayers. CaR mRNA (corrected with actin mRNA) was measured in freshly dispersed bovine parathyroid cells and at progressive time points in collagen and monolayer culture.

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Expression of CaR protein was examined in the pseudoglands by immunostaining. Results are shown in a representative image of an 18-day-old pseudogland (Fig. 8). Expression of the CaR is heterogeneous. No staining was seen when preimmune serum was used in place of the primary antibody.

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Figure FIG. 8.. CaR protein expression in pseudoglands. Expression of CaR protein was examined in bovine parathyroid pseudoglands by immunostaining. (A) A representative image of an 18-day-old pseudogland shows heterogeneous expression of the CaR. (B) No staining was seen when preimmune serum was used in place of the primary antibody.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The parathyroid glands play a central role in calcium homeostasis by monitoring the levels of extracellular calcium and secreting the appropriate amounts of PTH to maintain or to re-establish normal serum calcium. Elevated extracellular calcium has been reported to stimulate PTH peptide degradation, reduce PTH mRNA stability, and repress PTH gene transcription, although the latter observation has been recently challenged.(21, 22) Extracellular calcium can also prevent parathyroid gland proliferation. While much of our understanding of the cellular processes involved in the acute control of PTH secretion by calcium has come from studies in freshly isolated parathyroid cells, examination of the mechanisms involved in long-term control of parathyroid cell function by calcium has been hampered by the lack of an appropriate in vitro model. Primary cultures of bovine parathyroid cells rapidly lose their response to calcium, and no parathyroid cell lines that secrete PTH in a normally regulated fashion have been established.

Here, we report a novel and convenient model for studying long-term effects of calcium on parathyroid cells. Bovine parathyroid cells placed in a 3-D matrix of type I collagen will gradually break down the matrix and coalesce into a tight cellular mass, which we refer to as a pseudogland. The parathyroid pseudoglands retain both acute and chronic responses to calcium. The calcium set point, the calcium concentration that produces PTH secretion midway between the maximum and minimum rates, was 1.05 mM, similar to that of freshly dispersed adult bovine cells(23) and to the set point measured in vivo in normal individuals.(24) The response to calcium was stable over a period of several weeks. This may be attributed, in part, to the finding that the pseudoglands proliferate very slowly, if at all (no increase in DNA over 4 weeks). In this manner, the pseudoglands resemble normal parathyroid cells in vivo, which are known to rarely proliferate, as opposed to the more rapidly growing parathyroid cells in primary monolayer culture.

The loss of calcium responsiveness in primary cultures of bovine parathyroid cells, first reported by Nygren et al.,(25) was later associated with a decrease in CaR expression.(3, 4) However, the amount of CaR protein expressed in cultured parathyroid cells may not be the sole determinant of the calcium responsiveness. As shown in this study in the pseudoglands, the calcium response remained intact for several weeks in culture and then was lost, despite no change in CaR mRNA expression. Furthermore, although the CaR expression in the monolayers at 1 day in culture was decreased compared with dispersed cells, the calcium response was intact. The calcium response was subsequently lost by 1 week in monolayer culture, with no further decrease in CaR expression. This suggests that loss of the calcium response could be attributable to loss of factors other than loss of CaR expression, such as downstream signaling molecules.

Retention of calcium response in primary cultures of human parathyroid cells from uremic patients with secondary hyperparathyroidism has been reported by Roussanne et al.(6) The long-term response (5 months) of their parathyroid cells to calcium could be attributed to the growth characteristics of the hyperplastic uremic tissue used and/or to their culturing procedure that used a phosphate-rich culture medium and resulted in cells that generally aggregated into clusters. This latter feature lends further support to the notion that a more 3-D culture system favors retention of the calcium response in parathyroid cells. Using parathyroid tissue from patients with severe secondary hyperparathyroidism, however, may not result in a culture that is representative of normal parathyroid tissue, because the hyperplastic tissue often harbors cells with genetic abnormalities.(7) The use of bovine parathyroid tissue provides a source of normal parathyroid chief cells for study. In 1986, Ridgeway et al.(5) cultured bovine parathyroid cells as multicellular aggregates, termed organoids, using roller bottles. These organoids retained their response to extracellular calcium for at least 2 weeks in vitro. Why the organoids lost the response to calcium after that time is unknown, although they do note that necrotic areas could be detected after 1 week of culture in thicker parts of organoids formed from 4–5 × 106 cells. Elimination of the necrosis could be achieved by forming organoids from fewer cells (2-3 × 106). In our studies, each pseudogland is formed from 0.2 × 106 cells. The smaller mass of cells may allow better diffusion of medium nutrients to the innermost cells and prevent necrosis.

This study presents a novel method for culturing bovine parathyroid pseudoglands that produces a single, tight mass of cells that is easily manipulated: medium can be conveniently replenished; several pseudoglands can be incubated in a single well, thus conserving costly reagents; no specialized equipment is required; and the stable CaR expression and calcium responsiveness of the pseudoglands persists for several weeks, allowing for chronic studies. Calcium not only acutely suppresses PTH secretion in this model but also reduces PTH mRNA on a more chronic basis. Thus, this model provides a unique and convenient system in which to study the long-term effects of calcium on parathyroid gland function. The process(es) responsible for the coalescence of the parathyroid cells into pseudoglands and the factors that are involved in maintaining the calcium responsiveness are under investigation. The pseudogland model emphasizes the importance of 3-D structure in the proper functioning of parathyroid cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Patricia Clay for technical assistance in analyzing PTH. This work was supported by National Institutes of Health Grant DK-53774 (AJB).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
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  • 2
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  • 4
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  • 6
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  • 8
    Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, Macdonald PN, Brown AJ 1996 Phosphorus restriction prevents parathyroid gland growth-high phosphorus directly stimulates PTH secretion in vitro. J Clin Invest 97:25342540.
  • 9
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