Parathyroid Gland Hyperplasia in Renal Failure Part II
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Vitamin D Regulation of Uremia- and High Phosphate-Induced Parathyroid Cell Growth
Several laboratories have demonstrated a role for 1,25(OH)2D3 in suppressing parathyroid cell proliferation in vitro, and in vivo in uremia-induced PT hyperplasia in 5/6 nephrectomized rats.
As mentioned earlier, in several normal and transformed cell lines, the antiproliferative effects of 1,25(OH)2D3 involve the induction of the Cdk-inhibitor p21, through a transcriptional mechanism that requires the vitamin D receptor.
It is likely that this mechanism for 1,25(OH)2D3-induction of growth arrest also operates in the parathyroid glands.
An additional mechanism reported for 1,25(OH)2D3 suppression of parathyroid cell proliferation is by decreasing the expression of c-myc, an early replication-associated gene that regulates the progression from G1 to S phase in the cell cycle.
Resistance to vitamin D action, a common occurrence in end stage renal disease, limits the efficacy of therapy.
The causes for parathyroid resistance to vitamin D include a reduction in vitamin D receptor content in the parathyroid gland, and an impaired capacity of the 1,25(OH)2D3/VDR complex to regulate the expression of vitamin D responsive genes.
The latter persists even after correction of both serum 1,25(OH)2D3 and parathyroid vitamin D receptor content with vitamin D therapy. Since hyperphosphatemia is a major determinant of the resistance to vitamin D, preliminary studies examined the efficacy of vitamin D treatment [1,25(OH)2D3 and its analog 19-Nor-1,25(OH)2D2] in counteracting the mitogenic signals induced by early uremia and high dietary P.
Doses of 4 ng of 1,25(OH)2D3 and 30 ng of 19-nor-1,25(OH)2D2 were effective in controlling parathyroid hyperplasia with no hypercalcemic or hyperphosphatemic effects.
At these doses, both vitamin D compounds prevented the increase in PTH levels, mitotic activity and the enlargement of the parathyroid glands normally induced by uremia and worsened by high dietary P.
In this early-uremia rat model, the efficacy of 1,25(OH)2D3 and 19-nor-1,25(OH)2D2 in controlling both serum PTH and parathyroid gland growth was comparable to that described for P restriction.
Mechanistically, the suppression of uremic rat parathyroid cell growth by vitamin D treatment can also be partially accounted for enhanced expression of p21.
Studies in patients with secondary hyperparathyroidism support our findings in uremic rats for a role of increased p21 expression in parathyroid growth arrest.
The more aggressive nodular form of parathyroid hyperplasia coincided with the lowest p21 expression. Importantly, the lower the p21 content in human hyperplastic parathyroid glands, the lower the expression of the vitamin D receptor.
The findings from Tokumoto and collaborators and our own results on induction of parathyroid p21 after vitamin D therapy suggest that 1,25(OH)2D3 and 19-nor-1,25(OH)2D2 enhancement of parathyroid p21 expression is mediated by the vitamin D receptor, and could partially account not only for the antiproliferative effects of both sterols on high P-induced PT hyperplasia but for the enhanced growth associated with 1,25(OH)2D3 deficiency.
More importantly, in addition to inducing p21, treatment with 1,25(OH)2D3 or its analog 19-Nor-1,25(OH)2D2, prevented the increases in parathyroid TGFα and EGFR, induced by uremia and further enhanced by high dietary P.
This novel finding suggests that the control of parathyroid TGFα and EGFR expression by vitamin D treatment could also mediate the antiproliferative properties of 1,25(OH)2D3 and 19-nor-1,25(OH)2D2 on high-P induced parathyroid hyperplasia in renal failure.
Similar results were obtained when preventive vitamin D therapy (100 ng of the less calcemic vitamin D analog 19-nor-1,25(OH)2D2, three times a week, intraperitoneally), was given for two months starting after the onset of 5/6 nephrectomy, in rats fed a high P diet.
In these studies, the enlargement of the parathyroid glands was ameliorated by vitamin D therapy.
Importantly, this reduction in parathyroid growth was paralleled by a significant reduction in TGFα content.
This suggests an important role for the down-regulation of TGFα/EGFR signaling in vitamin D-antiproliferative action, even with prolonged exposure to the mitogenic stimuli by renal failure and high dietary P.
Interestingly, the induction of c-myc is one of the early events following activation of the conventional EGFR signaling pathway.
It is possible that in addition to the complex mechanisms mediating 1,25(OH)2D3/VDR inhibition of c-myc gene transcription, prevention of the increases in the parathyroid content of TGFα and EGFR by vitamin D also contributes to the decreased c-myc levels.
In summary, our studies provide the first evidence that the inhibition of parathyroid gland growth by vitamin D therapy is associated with both the induction of parathyroid p21 and the prevention of the increases in TGFα and its receptor, EGFR.
The interruption by vitamin D of the autocrine growth pathway involving TGFα and EGFR in parathyroid cells is a very efficient one, since it simultaneously downregulates the expression of ligand and receptor.
Exclusive downregulation of TGFα using antisense technology, while capable of reducing hyperproliferative activity in head and neck squamous carcinoma, was ineffective to arrest normal cell growth.
On the other hand, in human colon carcinoma, a marked inhibition of EGFR mRNA and protein and increased internalization of EGFR from the plasma membrane appear to mediate the antiproliferative effects of 1,25(OH)2D3.
Vitamin D downregulation of EGFR leads to reduced basal- and EGF mediated expression of cyclin D1 at both the mRNA and protein levels.
The relative contribution of this downregulation of TGFα or EGFR by vitamin D to parathyroid growth arrest is unknown.
The importance of 1,25(OH)2D3/vitamin D receptor–regulation of EGFR signaling is further supported by the demonstration of more aggressive growth patterns in colon carcinomas as the vitamin D receptor decreases.
Additional mechanisms could also contribute to the antiproliferative properties of 1,25(OH)2D3, as demonstrated in breast cancer cells.
In addition to inducing p21 and reducing c-myc expression, the inhibitory effects of 1,25(OH)2D3 on the cell cycle machinery controlling G1 to S transition, include the prevention of the activation of cyclin D1/cdk4 by mechanisms unrelated to the increase in p21.
1,25(OH)2D3 inhibits cdk2 by increased targeting of p21, decreased cyclin A and E association, and reduced phosphorylation of Rb.
Taken together these results suggest that the combined effects of 1,25(OH)2D3 deficiency and the resistance to vitamin D action in advanced renal failure, both worsened by hyperphosphatemia, could contribute to the switch from diffuse hyperplasia to the more aggressive nodular growth.
Potential mechanisms include the simultaneous enhanced co-expression of TGFα and EGFR in the parathyroid glands, as well as the reduction of parathyroid p21 and the additional 1,25(OH)2D3/VDR-dependent antimitogenic signals.
Identification of the molecular mechanisms mediating vitamin D downregulation of the TGFα/EGFR growth promoting activity in the hyperplastic parathyroid gland is an ongoing project in our laboratory that hopefully, will help design more effective strategies to arrest parathyroid growth.
Clinically, a more relevant question is whether vitamin D therapy is capable of suppressing parathyroid growth in established secondary hyperparathyroidism, specially in cases of concomitant hyperphosphatemia. To this end, 5/6 nephrectomized rats were fed a high P diet for two months to induce secondary hyperparathyroidism.
At this point, rats were divided in two groups and received either vehicle or the vitamin D analog 19-nor 1,25(OH)2D2, at a dose of 200 ng, intraperitoneally, three times a week, for two additional months.
While the parathyroid gland weight in the uremic rats receiving vehicle doubled between two and four months, no further increase in gland size was observed in the vitamin D treated uremic rats.
Parathyroid expression of markers of mitotic activity confirmed the efficacy of vitamin D therapy in arresting growth, thus preventing parathyroid gland enlargement.
The mechanisms mediating growth arrest, however, were not examined in these studies. Importantly, there were no signs of apoptosis in vitamin D treated rats indicating that there was prevention of further growth without regression of hyperplasia.
The recent design of vitamin D analogs with potent pro-apoptotic capabilities in several cell types raises hope for new therapeutic approaches for parathyroid hyperplasia.
Calcium Regulation of Uremia-Induced Parathyroid Growth
Further support for the pathophysiological relevance of changes in the expression of parathyroid p21, TGFα, and EGFR in controlling proliferative activity came from studies which evaluated the expression of these three proteins after the suppression of parathyroid hyperplasia by high dietary Ca or its further enhancement by low-Ca intake.
High dietary Ca controlled uremia-induced parathyroid hyperplasia as demonstrated by a reduction of both parathyroid gland size and the expression of two markers of mitotic activity, Ki and PCNA.
The mechanisms for high-Ca induction of p21 and prevention of the increases in TGFα and EGFR are unknown.
Studies in vitamin D receptor-ablated mice demonstrated the ability of a Ca-enriched diet to prevent the development of parathyroid hyperplasia in both hypocalcemic and normocalcemic states.
This clearly demonstrates that parathyroid growth arrest occurred through mechanisms independent of vitamin D receptor- or serum Ca levels.
Parathyroid p21 expression, however, was not assessed in these studies.
In relation to high-Ca control of the TGFα/ EGFR growth promoting signal, an association was recently reported between hypercalcemia and low plasma levels of TGFα in cancer patients, suggesting the possibility of systemic control of TGFα expression by Ca.
The persistence of such an association in renal failure patients, could partially explain the suppression of parathyroid growth by hypercalcemia.
The changes in p21, TGFα and EGFR in the parathyroid glands of uremic rats fed a high Ca diet suggest that increases in serum Ca or in intracellular Ca, induced by vitamin D therapy, could potentiate the effects of vitamin D itself in enhancing p21 expression and preventing the increases in TGFα and EGFR.
In contrast to the mechanisms for the antiproliferative effects of high dietary Ca, low-Ca intake exacerbated the parathyroid growth triggered by uremia by inducing TGFα and EGFR.
Conclusions
The molecular mechanisms regulating parathyroid hyperplasia in early uremia, according to our in vivo model.
The levels of co-expression of TGFα and EGFR, induced by uremia and further enhanced by either high dietary P or Ca restriction, correlate directly with increased proliferating activity and the enlargement of the parathyroid glands. Conversely, hypercalcemia, P restriction and vitamin D therapy, by preventing the increase in parathyroid TGFα and EGFR and simultaneous induction of p21, counteract the mitogenic signals and arrested gland growth.
The demonstration that the three main regulators of parathyroid growth, Ca, P and vitamin D modulate TGFα/EGFR signaling and p21 expression indicate the importance of these pathways in the pathogenesis of parathyroid hyperplasia and mark them as appropriate targets for more effective therapy.
