Parathyroid Gland Hyperplasia in Renal Failure Part I
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Introduction
Secondary hyperparathyroidism, a frequent complication of chronic renal failure, is characterized by parathyroid hyperplasia and enhanced synthesis and secretion of parathyroid hormone (PTH).
High circulating PTH levels are not only a major contributor to osteitis fibrosa and bone loss, typical features of renal osteodystrophy, but also to a variety of systemic defects including cardiovascular complications which increase mortality in renal failure patients.
A link between the mechanisms controlling proliferation and hormonal production also exists in normal parathyroid cells, which respond to the stimulus of chronic hypocalcemia not only by an increase in PTH release but with a secondary expansion in cell mass.
The mechanisms responsible for this link, however, remain poorly understood.
In renal failure, hypocalcemia, hyperphosphatemia and vitamin D deficiency are the three main direct causes of hyperparathyroidism.
Hyperphosphatemia and 1,25-dihydroxyvitamin D (1,25(OH)2D3) deficiency also enhance parathyroid function indirectly by lowering serum calcium (Ca).
The regulation of PTH synthesis by Ca, phosphate (P) and vitamin D has been extensively studied.
PTH-gene transcription is tightly controlled by Ca and 1,25(OH)2D3 through mechanisms that involve the calcium sensing receptor (CaSR) and the vitamin D receptor (VDR), respectively.
As renal disease progresses, a reduction in parathyroid content of both proteins renders the parathyroid glands more resistant to suppression of PTH synthesis and secretion in response to Ca and 1,25(OH)2D3.
Serum Ca and P levels also control PTH synthesis through post-transcriptional mechanisms that involve the binding of cytosolic proteins to the 3’-untranslated region of the PTH mRNA, thus regulating transcript stability and consequently, PTH translation rates.
Changes in the levels of serum Ca, P, 1,25(OH)2D3 and in the parathyroid content of the CaSR and the VDR also have a dramatic impact on parathyroid tissue growth.
However, the lack of an appropriate parathyroid cell line has precluded a better understanding of the pathogenic mechanisms mediating the potent effects of the three main regulators on the rate of parathyroid cell proliferation already induced by uremia.
This chapter summarizes the current understanding of parathyroid tissue growth and presents new insights, emerging from the 5/6 nephrectomized rat model, into the molecular mechanisms contributing to the regulation of parathyroid hyperplasia in early uremia by Ca, P and 1,25(OH)2D3.
Parathyroid Tissue Growth in Normal Conditions and in Renal Failure
The parathyroid gland is a low turnover, discontinuously replicating tissue composed of cells that rarely undergo mitoses.
Quiescent parathyroid cells, however, retain the potential to proliferate in response to the growth stimuli triggered by uremia, low Ca, high P or vitamin D deficiency.
As for most eukaryotic cells, the commitment of parathyroid cells to abandon quiescence (G0) and divide in response to growth stimuli depends on the net balance between the two opposing forces.
The forward force moving the cell through the phases of the cell cycle to complete a mitotic division is dictated by the activity of phase-specific complexes of cyclins and cyclin dependent kinases (Cdk).
The opposing force to suppress growth is exerted by a family of proteins, the Cdk- inhibitors, which bind cyclin/Cdk complexes inhibiting their activity.
Mitogenic signals triggered by uremia, low Ca, high P or vitamin D deficiency induce parathyroid cells to abandon quiescence and divide by increasing the activity of cyclin/Cdk complexes, decreasing Cdk-inhibitor threshold below the levels of cyclin/Cdk complexes or a combination of both.
In contrast, the antimitogenic signals elicited by high Ca, low P or vitamin D therapy could arrest cell growth by exerting the opposite effects, that is, decreasing the activity of cyclin/Cdk complexes, increasing Cdk-inhibitors to levels that exceed the amount of cyclin/Cdk complexes in the cell, or both.
The contribution of abnormalities in apoptotic rates to the enhanced parathyroid growth of uremia has also been examined.
The demonstration that the uremic state stimulates apoptosis, possibly as a compensatory reaction to the hyperproliferative activity, renders impaired apoptosis a highly improbable mechanism for the resultant hyperplasia. Different groups have demonstrated an association between parathyroid hyperplasia and changes in the content of cell cycle regulators.
Overexpression of PRAD1/cyclin D1, induced by a DNA re-arrangement of the PTH gene, is one genetic disorder in primary hyperparathyroidism.
The importance of cyclin D1 in parathyroid cell growth was conclusively demonstrated by the recent studies of Imanishi and coworkers using transgenic mice targeted to specifically overexpress cyclin D1 in the parathyroid gland.
These mice slowly develop large hyperplastic and in some cases adenomatous glands that expressed reduced CaSR.
This model mimics human parathyroid adenomas. In renal hyperparathyroidism, parathyroid glands initially grow diffusely and polyclonally.
Cells in the nodules then transform monoclonally and proliferate aggressively.
Neither the mechanisms triggering the initial increase in proliferative activity nor those resulting in changes in growth patterns are understood.
Changes in the expression of cell cycle components such as over-expression of cyclin D1 have been reported in patients with renal failure.
Studies by Tominaga and coworkers, comparing diffuse versus nodular hyperplasia, also demonstrated higher cyclin D1 expression in the latter.
However, different from parathyroid adenomas, there was no correlation between cyclin D1 and mitogenic activity in renal nodular hyperplasia.
These findings suggest that PRAD1/cyclin D1 overexpression induced by PTH gene re-arrangement may not be the major genetic abnormality responsible for tumorigenesis.
Heterogenous genetic changes appear to contribute to monoclonal proliferation of parathyroid cells induced either by the expression of PRAD1/cyclin D1 or by other mechanisms independent of the amplification of the protooncogene, such as reduction of the levels of a cell cycle repressor.
In fact, a reduction in the levels of the Cdk- inhibitor p27Kip1 has also been associated with hyperplastic growth.
The rapid de-differentiation of hyperplastic parathyroid cells in culture has precluded further assessment of the relative contribution of changes in Ca, P and vitamin D to the expression of components of the cell-cycle critical for growth control.
The experimental approach in identifying molecular mechanisms, critical in the design of more effective strategies for therapy, is therefore limited to the in vivo uremic rat model.
Dietary Phosphate Regulation of Parathyroid Cell Growth in Uremia
In contrast to the slow growth rate of human parathyroid adenomas, Naveh Many and collaborators demonstrated an increased rate of parathyroid cell proliferation fifteen days after 5/6 nephrectomy in rats.
In an identical time frame, uremia- induced mitotic activity was further enhanced by high dietary P but prevented by P restriction.
Furthermore, studies in our laboratory showed that most parathyroid growth induced by high dietary P (0.9 % P) occurred within five days of the onset of renal failure.
They also confirmed that, similar to human renal hyperparathyroidism, hyperplasia rather than hypertrophy is the main contributor to the enlargement of the parathyroid glands.
In contrast to the mitogenic effects of high P, dietary P restriction appears to counteract the proliferative signals induced by uremia, thus preventing parathyroid cell replication and consequently, the increase in parathyroid gland size.
The mechanism for the growth arrest by P restriction does not involve the induction of apoptosis.
Initial studies in rats were designed to identify the mechanisms mediating the opposing effects of dietary P manipulation on parathyroid cell growth in early renal failure.
These studies first examined the regulation of parathyroid expression of the Cdk-inhibitor p21 by dietary P.
The rationale for these studies was the well known effects of high dietary P to reduce and those of P-restriction to increase serum 1,25(OH)2D3 in normal individuals as well as in patients with mild and moderate renal failure.
1,25(OH)2D3, in turn, directly activates p21 gene transcription, an action mediated by the vitamin D receptor (VDR) as a transcriptional enhancer. 1,25(OH)2D3/VDR-induction of p21 expression is responsible for the potent antiproliferative properties of 1,25(OH)2D3 in cells of the monocyte-macrophage lineage, keratinocytes, and prostate cancer cell lines.
Furthermore, in contrast to the growth arrest promoted by increases in p21, the downregulation of p21 expression accounts for growth stimulation in the human epidermoid carcinoma cell line A431, human embryonic lung fibroblasts and glomerular epithelial cells.
These reports led us to hypothesize that opposite changes in parathyroid p21 content induced by dietary P manipulations, either directly or through changes in 1,25(OH)2D3, could contribute to the potent inhibition or stimulation of mitotic activity.
The results of these studies demonstrated that, indeed, the low P- induction of p21 mRNA and protein content in the parathyroid glands contributes to the antiproliferative effects of P-restriction on uremia-induced parathyroid cell growth.
P-restriction decreased serum P and induced a two-fold increase in parathyroid p21 mRNA levels as soon as two days after 5/6 nephrectomy.
This modest increase in p21 mRNA in P-restricted rats, remained 2-fold higher than in the high P group through day 7, and resulted in a substantially higher expression of p21 protein.
The latter finding suggests the existence of an additional post-transcriptional mechanism for P restriction in the up-regulation of p21 expression in rat parathyroid glands. Although this hypothesis is difficult to test using our in vivo model, a similar induction of p21 expression through post-transcriptional regulatory mechanisms was reported both in fibroblasts and murine erythroleukemia cells.
Importantly, in contrast to our working hypothesis, low P induction of parathyroid p21 was not the result of an increase in serum 1,25(OH)2D3 concentrations. Clearly, pathways other than the 1,25(OH)2D3-vitamin D receptor axis mediate both low-P activation of p21 gene transcription and the post-transcriptional enhancement of p21 protein expression.
The induction of p21 is sufficient to induce growth arrest in monocyte-macrophages, keratinocytes, and human cancer cells and to suppress tumorigenicity in vivo.
In our uremic rat model, a role for low-P induction of p21 expression in the arrest of parathyroid cell growth is supported by the demonstration that temporal increases in p21 protein expression correlate inversely with parathyroid levels of the marker of mitotic activity proliferating nuclear cell antigen (PCNA).
Intestinal growth as well as p21 and PCNA content remained unchanged with dietary-P manipulation, thus supporting the specificity for the parathyroid glands of the antimitogenic effects of P restriction in these uremic rats.
The mechanisms by which p21 arrests growth. p21 is a member of the family of cdk-inhibitors that includes p27 and p57. p21 inhibits multiple cyclin dependent kinases including cdk1, cdk2, cdk4 and cdk6.
As seen in the left panel, p21 is also a component of a multiprotein complex which includes cyclins, cdk, and PCNA.
At a stoichiometry of one molecule of p21 per complex, this multicomplex phosphorylates the retinoblastoma (Rb) protein resulting in the release of E2F for autoactivation and activation of S-phase genes (DNA replication phase) to complete a mitotic division.
As depicted in the right panel, the induction of p21 in response to various stimuli, results in inhibition of G1-cyclin/cdk complexes and G1 growth arrest. This effect has been related, at least in part, to excess p21 blocking activation of G1-cyclin/cdk complexes by cdk7/cyclin H, thus preventing both Rb-phosphorylation and E2F activation. p21 has also been shown to inhibit replication by binding to PCNA trimers causing DNAse–polymerase to lose processivity.
In contrast to our initial hypothesis, high-dietary P had no detectable effect on parathyroid expression of p21 mRNA or protein.
Thus, reduction in intracellular p21 is not the cause for the mitogenic properties of high P in uremic rat parathyroid glands.
In the search for mitogenic stimuli triggered by high dietary P, we next focused on transforming growth factor-α (TGFα).
TGFα, known to promote growth not only in malignant transformation but also in normal tissues, is enhanced in hyperplastic and adenomatous human parathyroid glands.
Immunohistochemical quantitation of TGFα content in the parathyroid glands of uremic rats showed the pattern of high TGFα expression reported for human hyperplastic glands.
Furthermore, as depicted in
TGFα expression peaked by day 5 and remained higher than in the low-P group through day 7.
The increases in TGFα induced by high P paralleled those in PCNA expression and were specific for the parathyroid glands since there were no changes in intestinal growth or TGFα content.
Thus, high-P induction of TGFα in the uremic rat parathyroid gland may constitute an autocrine signal which further stimulates uremia-induced parathyroid cell proliferation.
Additional support for induction of parathyroid TGFα to mediate the mitogenic properties of high dietary P came from studies in rats one month after 5/6 nephrectomy.
The high parathyroid TGFα decreased to levels found in normal controls within three days after reducing dietary P intake from 0.9% to 0.2%.
The rapid return of parathyroid TGFα content to normal levels by P restriction also suggests that low P may counteract uremia-induced parathyroid cell growth not only through induction of p21 expression, but also by preventing the enhancement of parathyroid TGFα.
The mature, soluble form of TGFα is produced from double proteolytic cleavage of a transmembrane precursor.
Both membrane anchored and soluble forms of TGFα signal through the epidermal growth factor receptor (EGFR), which is normally expressed in the parathyroid glands.
Increases in parathyroid TGFα could induce cell growth through autocrine and paracrine mechanisms upon activation of the EGFR by the mature TGFα isoform, and, as demonstrated in other tissues, through a less characterized juxtacrine pathway involving the transmembrane TGFα isoform from an adjacent parathyroid cell.
The EGFR is a 170 KD membrane glycoprotein with an extracellular-ligand binding domain, a short transmembrane helix and an intracellular domain which has tyrosine kinase activity.
Ligand binding induces receptor dimerization and simultaneous activation of its intrinsic tyrosine kinase.
Upon activation, EGFR signals to the nucleus mainly through the Ras/ mitogen-activated protein kinase (MAPK).
Following MAPK activation, cyclin D1 is induced and drives the cell cycle from the G1 to the S phase.
In addition to this signal transduction pathway typical for cell membrane receptors, a role for nuclear EGFR as a transcription factor has recently been reported.
The in vivo demonstration of nuclear EGFR associated with adenosine-thymidine-rich regions in the cyclin D1 promoter may partially explain the strong correlation between nuclear EGFR localization and high proliferating activity in several tissues.
In human parathyroid glands, Gogusev and coworkers demonstrated the presence of EGFR protein in 4 out of 5 adenomas, in 13 of 15 tissue samples of hyperplasia secondary to renal failure, and in most samples of normal parathyroid tissue.
No differences in the expression patterns were observed among groups.
However, studies in 104 human hyperplastic parathyroid glands, which failed to detect EGFR protein, showed higher EGFR mRNA expression in carcinoma and primary hyperplasia compared to adenomas and hyperplasia secondary to renal failure.
The strong association between high proliferative activity and nuclear EGFR localization could partially account for the discrepancy between protein and mRNA expression in the latter studies.
The concept that co-expression of TGFα and EGFR could contribute to non neoplastic endocrine hyperplasia led us to examine the dietary-P regulation of parathyroid EGFR expression in rat parathyroid glands.
These findings indicate that the induction of parathyroid co-expression of TGFα and its receptor, EGFR, by uremia, acts as a mitogenic signal which is further enhanced by high dietary P, can be prevented by P restriction and counteracted through induction of p21.
These are novel insights into the molecular mechanisms associated with the potent opposing effects of high and low P regulation of parathyroid growth.
They suggest that, in addition to P restriction or the use of P binders, therapeutic maneuvers oriented to further induce p21 and inactivate TGFα/EGFR growth-promoting signals could be more effective in slowing the progression of secondary hyperparathyroidism.
Despite this important step forward, the most critical target to optimize therapy, the parathyroid sensing mechanism for changes in extracellular inorganic P, remains unknown.
The assessment of the efficacy of 1,25(OH)2D3 in enhancing parathyroid p21 to levels capable of counteracting the mitogenic signals triggered by uremia and high dietary P was the next step in our research.
