Oestrogen deficiency--does it have a role in the genesis of skeletal problems in dialysed women?
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Biomedical subjects
T Naveh-Many
Publications and source records attributed to T Naveh-Many.
Parathyroid hormone mRNA levels are increased by progestins and vary during rat estrous cycle.
Estrogen increases parathyroid hormone (PTH) mRNA levels in vivo in ovariectomized rats. We now show that the 19-norprogestin R-5020 given to weanling rats or mature ovariectomized rats led to a twofold increase in thyroparathyroid PTH mRNA levels. This increase in PTH mRNA occurred at 24 and 48 h after progesterone but not at 72 h. There were no changes in serum calcium. In vitro, in primary cultures of bovine parathyroid cells, progesterone increased PTH mRNA levels threefold at 10(-8) M and twofold at 10(-9) M after 24 h. Progesterone receptor (PR) mRNA was demonstrated in rat parathyroid tissue by in situ hybridization and in human parathyroid adenoma by immunohisto-chemistry. Changes in PTH mRNA levels during the rat estrous cycle were also studied. At proestrus and estrus PTH mRNA levels were increased significantly by three- and fourfold compared with diestrus. Our results confirm that the parathyroid gland is a target organ for the ovarian sex steroids estrogen and progesterone and are of physiological relevance as shown by the changes during estrus.
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Regulation of parathyroid hormone messenger RNA levels by protein kinase A and C in bovine parathyroid cells.
Secretion of parathyroid hormone (PTH) is regulated by Ca2+ as well as by protein kinases A and C. In this study we report that protein kinases A and C regulate PTH messenger RNA levels in vitro in dispersed bovine parathyroid cells. Incubation of bovine parathyroid cells with cholera toxin (10(-9) M), which activates adenylate cyclase and indirectly stimulates protein kinase A, increased PTH mRNA levels about 2-fold after 3 and 7 h incubation, but not at 24 h. Incubation with pertussis toxin (5 x 10(-9) M), which blocks the high-calcium-mediated inhibition of cyclic adenosine monophosphate accumulation in these cells, also reversed the inhibition of PTH mRNA levels at high Ca2+ (2.0 mM) with a marked increase in PTH mRNA levels. Pertussis toxin also increased PTH mRNA at a low extracellular Ca2+ concentration (0.7 mM) (4-fold increase) and a normal concentration (1.25 mM) (2-fold increase). Inhibition of protein kinase C both by staurosporine (1 x 10(-8) M) and by prolonged incubation with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (1 x 10(-7) M), decreased PTH mRNA levels at 24 h, reaching approximately 40% and 5% of control, respectively. Staurosporine and PMA had no effect on PTH mRNA levels at 3 h. The inactive phorbol ester, phorbol 12-13-dibutyrate (PDBu), had no effect on PTH mRNA levels at 1 and 24 h. There were no changes in a control gene 18S RNA in these studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylate Cyclase Toxin↗
Oestrogens and calcium regulatory hormones: potential implications for bone.
Postmenopausal oestrogen deficiency is associated with the development of osteoporosis. Oestrogen therapy prevents further bone loss but does not have an anabolic effect. The only treatment with an anabolic effect on bone is intermittent parathyroid hormone treatment. Oestrogens have a direct action on the parathyroid to increase parathyroid hormone gene expression and parathyroid hormone secretion. They exert this effect at doses that are too low to cause the uterotrophic effect of oestradiol. Osteoporotic patients have a decreased parathyroid hormone secretory response to changes in serum calcium, supporting the experimental data that oestrogens have a direct effect on the parathyroid. The value of parathyroid hormone treatment is limited by the need for parenteral therapy. The ability of oestrogens to increase parathyroid hormone secretion suggests that the intermittent administration of oestrogen analogues, at doses that exert no effects on breast tissue and the uterus, would be the optimal treatment for osteoporosis.
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Coordinate regulation of rat renal parathyroid hormone receptor mRNA and Na-Pi cotransporter mRNA and protein.
Parathyroid hormone (PTH) acts on the kidney by binding to the PTH receptor, leading to a decrease in the active renal reabsorption of phosphate by the Na-Pi cotransporter, which is also independently activated by hypophosphatemia. We have studied the effects of hypo- and hyperparathyroidism and hypophosphatemia on PTH receptor mRNA and Na-Pi cotransporter mRNA and protein. Both surgical parathyroidectomy and hypophosphatemia, which itself leads to hypoparathyroidism, led to an upregulation of the PTH receptor mRNA and Na-Pi cotransporter mRNA and protein. Parathyroidectomized rats fed a low-Pi diet had an increase in PTH receptor and Na-Pi cotransporter mRNAs. Diet-induced hyperparathyroidism had no effect on PTH receptor mRNA and Na-Pi cotransporter mRNA and protein. The effect of hypoparathyroidism and hypophosphatemia to increase both PTH receptor mRNA and Na-Pi cotransporter mRNA and protein shows that there is a tight coordinate regulation of these factors, which are both involved in PTH action.
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Parathyroid hormone gene expression in hypophosphatemic rats.
Phosphate is central to bone metabolism and we have therefore studied whether parathyroid hormone (PTH) is regulated by dietary phosphate in vivo. Weanling rats were fed diets with different phosphate contents for 3 wk: low phosphate (0.02%), normal calcium (0.6%), normal phosphate (0.3%), and calcium (0.6%); high phosphate (1.2%), high calcium (1.2%). The low phosphate diet led to hypophosphatemia, hypercalcemia, and increased serum 1,25(OH)2D3 together with decreased PTH mRNA levels (25 +/- 8% of controls, P < 0.01) and serum immunoreactive PTH (4.7 +/- 0.8: 22.1 +/- 3.7 pg/ml; low phosphate: control, P < 0.05). A high phosphate diet led to increased PTH mRNA levels. In situ hybridization showed that hypophosphatemia decreased PTH mRNA in all the parathyroid cells. To separate the effect of low phosphate from changes in calcium and vitamin D rats were fed diets to maintain them as vitamin D-deficient and normocalcemic despite the hypophosphatemia. Hypophosphatemic, normocalemic rats with normal serum 1,25(OH)2D3 levels still had decreased PTH mRNAs. Nuclear transcript run-ons showed that the effect of low phosphate was posttranscriptional. Calcium and 1,25(OH)2D3 regulate the parathyroid and we now show that dietary phosphate also regulates the parathyroid by a mechanism which remains to be defined.
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Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D.
Secondary hyperparathyroidism is characterized by an increase in parathyroid (PT) cell number, and parathyroid hormone (PTH) synthesis and secretion. It is still unknown as to what stimuli regulate PT cell proliferation and how they do this. We have studied rats with dietary-induced secondary hyper- and hypoparathyroidism, rats given 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and rats after 5/6 nephrectomy for the presence of PT cell proliferation and apoptosis. PT cell proliferation has been measured by staining for proliferating cell nuclear antigen (PCNA) and apoptosis by in situ detection of nuclear DNA fragmentation and correlated with serum biochemistry and PTH mRNA levels. A low calcium diet led to increased levels of PTH mRNA and a 10-fold increase in PT cell proliferation. A low phosphate diet led to decreased levels of PTH mRNA and the complete absence of PT cell proliferation. 1,25 (OH)2D3 (25 pmol/d x 3) led to a decrease in PTH mRNA levels and unlike the hypophosphatemic rats there was no decrease in cell proliferation. There were no cells undergoing apoptosis in any of the experimental conditions. The secondary hyperparathyroidism of 5/6 nephrectomized rats was characterized by an increase in PTH mRNA levels and PT cell proliferation which were both markedly decreased by a low phosphate diet. The number of PCNA positive cells was increased by a high phosphate diet. Therefore hypocalcemia, hyperphosphatemia and uremia lead to PT cell proliferation, and hypophosphatemia completely abolishes this effect. Injected 1,25 (OH)2D3 had no effect. These findings emphasize the importance of a normal phosphate and calcium in the prevention of PT cell hyperplasia.
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New aspects in the control of parathyroid hormone secretion.
Ca2+ binds to a parathyroid cell Ca2+ receptor, which is G protein-coupled and activates inositol triphosphate production. Mutations in the Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Chronic hypocalcemia increases parathyroid hormone messenger RNA levels and parathyroid cell hyperplasia. Parathyroid cells in vitro are heterologous in their response to Ca2+. The concept of a higher Ca2+ set-point in secondary hyperparathyroidism is controversial. Calcitriol is more effective than the less hypercalcemia analogues in decreasing parathyroid hormone messenger RNA and immunoreactive parathyroid hormone levels, and its kinetics are well established. Phosphate and estrogens regulate the parathyroid independently of 1,25 dihydroxyvitamin D3 and Ca2+. The physiology of the effects of endothelin and insulin-like growth factors on the parathyroid need to be established. Important advances are being made in understanding the regulation of parathyroid hormone synthesis and secretion, which are relevant to both normal physiology and the pathogenesis and treatment of diseases such as the secondary hyperparathyroidism of renal failure and osteoporosis.
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Regulation of parathyroid hormone synthesis and secretion.
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Diminished calcitonin secretion after ovariectomy without apparent reduction in calcitonin content in the rat.
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Calcitonin gene regulation in vivo.
In view of the importance of the caliotropic hormones to calcium homeostasis and bone strength we have studied the regulation of the expression of the calcitonin gene in vivo in the rat. Normal rats were injected with 1,25-dihydroxyvitamin D3 (12.5-200 pmol/100 g body weight) and calcitonin mRNA levels measured. 1,25(OH)2D3 led to a marked decrease in calcitonin mRNA levels, which reached 4% of basal at 24 h. Nuclear transcript experiments showed that the effect was transcriptional. After large changes in serum calcium for periods from 6 h to 3 weeks there were no differences in calcitonin mRNA levels. Estrogens given to ovariectomized rats led to increases in calcitonin mRNA. The estrogen receptor was present in the thyroid C-cells. Therefore the calcitonin gene is regulated by vitamin D and estrogens, but not by calcium.
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Effects of calcitriol, 22-oxacalcitriol, and calcipotriol on serum calcium and parathyroid hormone gene expression.
Calcitriol markedly decreases PTH gene transcription, but because of the concern about hypercalcemia, there is interest in nonhypercalcemic analogs. We have studied the effects of calcitriol, oxacalcitriol, and calcipotriol on serum calcium and PTH mRNA levels in vivo and in vitro. In vivo in rats, calcitriol was the most effective analog in decreasing PTH mRNA levels, with a maximal effect of about 70% at 25-100 pmol after 24 h. Only 100 pmol led to hypercalcemia. Oxacalcitriol led to a maximal decrease in PTH mRNA levels of 44% at 2 and 5 nmol, similar to 1 nmol calcipotriol. Oxacalcitriol at 200 and 500 pmol led to an increase in serum calcium at 3 h, but not at 6 and 24 h, unlike calcitriol (100 pmol) which caused an increase only at 24 h. In vitro, in primary cultures of bovine parathyroid cells, calcitriol and oxacalcitriol both decreased PTH mRNA levels at similar concentrations. Therefore, in vivo calcitriol is the most effective analog studied in decreasing PTH mRNA levels, including a range of doses that does not cause hypercalcemia. Oxacalcalcitriol and calcipotriol are less effective, but have a wider dose range where they do not cause hypercalcemia. The results in vitro confirm that oxacalcitriol and calcitriol both effectively decrease PTH mRNA levels at the same concentration. The marked activity of calcitriol analogs in vitro compared to in vivo probably reflects differences from calcitriol in their pharmacokinetics.
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Regulation of calcitonin gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat.
High calcium leads to the secretion of calcitonin, and the administration of 1,25-dihydroxyvitamin D3 leads to a decreased transcription of the calcitonin gene. We now report the effect of chronic hypercalcemia, hypocalcemia, and vitamin D deficiency on calcitonin gene expression in vivo in the rat. Hypercalcemia was created by calcium infusions for 6 h, a high-calcium diet given to weanling rats for 3 weeks, and the transplantation of the Walker carcinosarcoma 256 cell line. Despite serum calcium as high as 22 mg/dl, there was no difference in calcitonin mRNA levels among these rats. The control genes studied, actin and somatostatin, which is specific for C cells in the thyroparathyroid tissue, also did not differ among the different groups of rats. Injected 1,25-(OH)2D3 decreased calcitonin mRNA levels at 6 h, as previously reported. Hypocalcemia, created by feeding diets deficient in calcium and vitamin D to weanling rats for 3 weeks, had no effect on calcitonin mRNA levels, in contrast to the large increases in PTH mRNA levels. These results demonstrate that calcitonin gene expression in vivo in the rat is regulated by administered 1,25-(OH)2D3 but not by changes in serum calcium.
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Estrogen receptors and biologic response in rat parathyroid tissue and C cells.
The expression of the PTH and calcitonin genes is dramatically decreased by 1,25(OH)2D3 in vivo, and the PTH gene expression is increased by hypocalcemia. We have now studied the effect of estrogens on the expression of these genes in vivo. 17 beta-Estradiol, given to ovariectomized rats, led to a fourfold increase in PTH mRNA and calcitonin mRNA levels. These effects occurred 24 h after single injections of 37-145 nmol estradiol, or after constant infusions of 12 pmol/d for 1 or 2 wk, where there was no effect on serum calcium levels. The estrogen receptor mRNA was demonstrated in the thyroparathyroid tissue by polymerase chain reaction. The estrogen binding was localized to the parathyroid and C cells by immunohistochemistry. Uterus weight was increased by repeated larger doses (73 nmol/d x 7) of estradiol, but not by the small doses (12 pmol/d for 1 or 2 wk) which were effective on the PTH and calcitonin genes, suggesting a sensitive endocrine effect. These results confirm that the parathyroid and C cells are target organs for estrogen, leading to an increased expression of PTH and calcitonin, which by their combined anabolic effect on bone would help prevent osteoporosis.
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Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat.
In vivo in the rat 1,25(OH)2D3 decreases and a low calcium increases PTH mRNA levels. We now report the effect of 3 and 8 wk of changes in dietary vitamin D and calcium on PTH mRNA levels. PTH mRNA levels were increased by 3 wk of calcium deficiency (five times), a vitamin D-deficient diet (two times), and combined deficiency (10 times), but not changed by high calcium. Vitamin D-deficient-diet rats' PTH mRNA did not decrease after a single large dose of 1,25(OH)2D3, but did decrease partially after repeated daily doses of 1,25(OH)2D3. Rats after a vitamin D-, calcium-deficient (-D-Ca) diet did not respond to changes in serum calcium at 1 h. Flow cytometry of isolated cells from parathyroid-thyroid tissue separated the smaller parathyroid from the larger thyroid cells and allowed an analysis of parathyroid cell number. In normal vitamin D/normal calcium (NDNCa) rats the parathyroid cells were 24.7 +/- 3.4% (n = 6) of the total cell number, whereas in -D-Ca rats they were 41.8 +/- 6.6% (n = 6) (P less than 0.05). That is, -D-Ca rats had 1.7 times the number of cells, whereas they had 10 times the amount of PTH mRNA, indicating the major contribution (6 times) of increased PTH gene expression per cell. Moreover, a calcium-deficient, more so than a vitamin D-deficient diet, amplifies the expression of the PTH gene, and vitamin D is necessary for an intact response of PTH mRNA to 1,25(OH)2D3 or calcium.
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Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by 1,25-dihydroxyvitamin D3 in the parathyroid in vivo.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3 dramatically decreases parathyroid hormone (PTH) gene transcription. We have now studied the effect of 1,25(OH)2D3 on the 1,25(OH)2D receptor (VDR) in the parathyroid in vivo. Rats were injected with 1,25(OH)2D3 and the parathyroid-thyroid tissue analyzed for PTHmRNA and VDRmRNA. 1,25(OH)2D3 (50 and 100 pmol ip) decreased PTHmRNA at 6 h with a maximum at 48 h (less than 4% of basal), whereas VDRmRNA was increased only after 6 h with a 1.7-fold increase at 24 h. VDRmRNA levels peaked at 25 pmol 1,25(OH)2D3 with a twofold increase. Serum calcium did not affect VDRmRNA. Parathyroid VDRmRNA ran at 2.2 and 4.4 kb, whereas duodenum VDRmRNA had a single band, all of which increased after 1,25(OH)2D3. Weanling rats on a vitamin D-deficient diet for 3 wk had a more intense 2.2-kb transcript, whereas vitamin D-replete rats had a more intense 4.4-kb band. Dispersed parathyroid-thyroid cells were separated by a flow cytometry (FACS) into a parathyroid cell peak containing PTHmRNA and a second peak with cells positive for thyro-globulin mRNA and calcitonin mRNA. VDRmRNA was concentrated in the parathyroid cell peak. In situ hybridization of parathyroid-thyroid and duodenum for VDRmRNA showed its localization to the parathyroid cells and the duodenal mucosa. Therefore, the VDRmRNA in the parathyroid-thyroid tissue represents predominantly parathyroid cell and not C-cell VDRmRNA which is also a 1,25(OH)2D3 target organ. The increased VDR gene expression in the parathyroid cell would amplify the effect of 1,25(OH)2D3 to decrease PTH gene transcription.
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Regulation of parathyroid cell gene expression in experimental uremia.
The secondary hyperparathyroidism of renal failure is an important component of renal osteodystrophy. We studied PTHmRNA levels and their regulation in control and subtotal nephrectomized (5/6 NX) rats at 3 wk, as well as levels of the 1,25(OH)2D3 receptor mRNA in parathyroids. Serum 1,25(OH)2D levels were decreased in 5/6 NX, whereas PTHmRNA levels were increased (7 +/- 0.7 OD U, N = 4) compared to controls (2.1 +/- 1.2, P less than 0.01); both decreased after 1,25(OH)2D3 (100 pmol/100 g body weight). Similar results were found in 5/6 NX rats after 3 months. There was no change in actin mRNA levels. PTHmRNA levels were highest in 5/6 NX rats with the most severe renal failure. The parathyroid gland 1,25(OH)2D3 receptor mRNA levels were not different between 5/6 NX rats and controls and were not affected by 1,25(OH)2D3 (100 pmol/100 g body weight daily) at 1 or 3 days. PTHmRNA levels of 5/6 NX rats did not increase when the serum calcium was decreased from 2.8 +/- 0.05 mmol/L to 0.9 +/- 0.15 mmol/L at 3 or 5 h, which contrasted with the marked increase in PTHmRNA in normal rats after hypocalcemia. As in normal rats, after hypercalcemia (4.8 mmol/L at 1 h) there was no change in the 5/6 NX rats' PTHmRNA levels. These results show that 5/6 NX rats have increased PTHmRNA levels that are normally regulated by injected 1,25(OH)2D3 but not by calcium. Parathyroid gland 1,25(OH)2D receptor mRNA levels are not increased in 5/6 NX in contrast to the increased PTHmRNA, which reflects the larger glands of uremia. 1,25(OH)2D receptor mRNA levels were not regulated by 1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)
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Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitamin D.
In vivo 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) decreased PTH and calcitonin gene transcription. A low calcium is the major signal for PTH secretion, and a high calcium for calcitonin secretion. We report here that calcium has no effect on calcitonin messenger RNA (mRNA) levels in vivo in the rat, but that a low calcium markedly stimulates PTH mRNA levels. Serum calcium was decreased by ip phosphorus and increased by calcium gluconate (ip or iv infusion) and demonstrated that a low serum calcium markedly increased PTH mRNA levels whereas a high serum calcium had no effect. There was no change in mRNAs for calcitonin or actin in the same thyroparathyroid extracts. After phosphorus ip serum calcium decreased from 10.4 to 8.5 mg/dl and PTH mRNA increased up to 3-fold at 1, 3, and 6 h. Gel blots showed that a low calcium increased PTH mRNA levels with no change in its size (833 base pairs). Calcitonin ip decreased both serum calcium and phosphorus with an up to 5-fold increase in PTH mRNA at 1 h, thus demonstrating that the effect of phosphorus on PTH mRNA was due to the low serum calcium and not the high serum phosphorus. When phosphorus and 1,25(OH)2D3 (100 pmol/100 g) were injected together, despite the low serum calcium, there was a decrease in PTH mRNA levels. These results show a linear relationship between calcium and PTH gene expression, but not calcitonin or actin, with a dominant role for 1,25(OH)2D3.
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