Biomedical subjects
R Rizzoli
Publications and source records attributed to R Rizzoli.
Cyclic AMP increases the release of parathyroid hormone-related protein from a lung-cancer cell line.
Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of malignant hypercalcemia by stimulating bone resorption and/or renal tubular reabsorption of calcium. In cultured cancer cells, its production can be influenced by various factors or ions, but the regulation of its production is still poorly understood. We investigated the effects of stimulators of cAMP synthesis on PTHrP release by a human lung squamous-carcinoma cell line (BEN). In superfused cells grown on microcarrier beads, PTHrP production was significantly increased after incubation with calcitonin for only 20 min. The release of immunoreactive and bioactive PTHrP was increased by incubating the cells with forskolin, 3-isobutyl-1-methylxanthine or dibutyryl cAMP even in the presence of the protein-synthesis inhibitor cycloheximide for 6 hr. The calcitonin-mediated stimulation was not accompanied by concomitant changes in PTHrP mRNA. The microfilament-disrupter cytochalasin D was shown to enhance the basal and calcitonin-induced production of PTHrP. These results indicate that stimulators of cAMP synthesis enhanced PTHrP release by BEN cells.
Effects of treatment of malignancy-associated hypercalcemia on serum parathyroid hormone-related protein.
Parathyroid hormone-related protein (PTHrP) is the primary mediator of hypercalcemia in patients with malignancy-associated hypercalcemia. We conducted this study to examine the effects of treatment with a bisphosphonate on serum PTHrP. We analyzed 41 episodes of hypercalcemia occurring in 38 patients: 22 patients received alendronate, and 16 were treated with pamidronate. At baseline, 29 patients had an increased serum PTHrP (group I) and 9 had low or undetectable levels (group II). The two groups did not differ significantly in baseline hypercalcemia (3.26 versus 3.41 mM) or the response of serum calcium to therapy. Serum calcium was normalized in 88% of group I and 70% of group II patients. Lowering of the mean calcium level was not associated with a change in the level of PTHrP in group I patients (40.2 versus 36.7 pgEq/ml) or group II patients. We also analyzed data on serum PTH and 1,25-(OH)2D in 20 of the patients. Serum PTH rose with treatment in group I patients (9.7-40.2 pg/ml, p < 0.05), as did the serum 1,25-(OH)2D (19.1-32.4 pg/ml, p < 0.001). Similarly, treatment of group II patients was associated with an increase in serum PTH (9.8-37.2 pg/ml) and serum 1,25-(OH)2D (22.9-40.2 pg/ml). The individual increases in 1,25-(OH)2D levels associated with therapy could not be predicted from the level of PTHrP or the changes in levels of serum calcium or PTH. Our data show that effective treatment of malignancy-associated hypercalcemia is not associated with a consistent change in serum levels of PTHrP. Therapy is associated with a variable increase in the serum levels of PTH and 1,25-(OH)2D.
Effects of epidermal growth factor on parathyroid hormone-related protein production by mammary epithelial cells.
Parathyroid hormone-related protein (PTHrP) plays a prominent role in the pathogenesis of humoral hypercalcemia of malignancy. However, it is also expressed in various nonmalignant tissues, particularly during fetal organogenesis and tissue differentiation. Thus, PTHrP is synthesized in skin, placenta, and mammary gland during lactation. Little is known, however, about the regulation of PTHrP synthesis and release in nontumoral cells. We investigated the regulation of PTHrP production by epidermal growth factor (EGF), a factor of major importance in the development of lactating breast, in primary cultures of rat mammary epithelial cells. EGF stimulated the production of immunoreactive and bioactive PTHrP in a time- and concentration-dependent manner. A 12 h incubation with 10 ng/ml of EGF increased PTHrP production by 36.0 +/- 7.1% (n = 7 experiments, p < 0.01). This was accompanied by an increase in PTHrP mRNA steady-state levels. The production of PTHrP was stimulated by the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) by 82.9 +/- 9.7% (n = 4 experiments, p < 0.01). The effects of PMA and EGF were additive. The EGF-induced stimulation appeared to be independent of extracellular calcium concentration, prostaglandin, or cAMP synthesis, but may have involved tyrosine kinase-mediated mechanisms. These results indicate that EGF was capable of increasing the production of PTHrP by cultured mammary epithelial cells. They also suggest that factors activating the PKC pathway are involved in the upregulation of PTHrP expression in mammary epithelial cells.
High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.
HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.
Longitudinal measurement of regional and whole body bone mass in young healthy adults.
The so-called peak bone mass (PBM) represents the highest amount of bony tissue achieved during life at a given site of the skeleton. It has been suggested that PBM might be achieved as late as the fourth decade, but recent data have indicated that PBM is already achieved by the end of sexual maturation, namely at the end of the second decade. The solving of this apparent controversy is of interest for a better understanding of bone homeostasis and for defining the cohort of normal subjects to be evaluated in order to establish a PBM reference range--necessary for the diagnosis of osteoporosis and evaluation of the fracture risk. To study bone mass evolution in young healthy adults and to determine whether such a cohort can be used to establish PBM reference values, we measured bone mineral density (BMD) in sixty 20- to 35-year-old young healthy adults by dual-energy X-ray absorptiometry at the levels of the lumbar spine (in both anteroposterior and lateral views), femoral neck, trochanter region, total hip and of Ward's triangle, as well as whole-body BMD and bone mineral content (BMC) in cross-sectional and longitudinal studies. In the cross-sectional analysis, none of the bone mass variables was dependent on age using linear regression analysis. The longitudinal study indicated that the mean changes in lumbar spine, proximal femur and whole body BMD or BMC determined after a 1-year interval were not statistically different from zero in either females or males aged 20-35 years.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of calcium supplements on femoral bone mineral density and vertebral fracture rate in vitamin-D-replete elderly patients.
The efficacy of calcium (Ca) in reducing bone loss is debated. In a randomized placebo-controlled double-masked study, we investigated the effects of oral Ca supplements on femoral shaft (FS), femoral neck (FN) and lumbar spine (LS) bone mineral density (BMD), and on the incidence of vertebral fracture in vitamin-D-replete elderly. Ninety-three healthy subjects (72.1 +/- 0.6 years) were randomly allocated to three groups receiving 800 mg/day Ca in two different forms or a placebo for 18 months. Sixty-three patients (78.4 +/- 1.0 years) with a recent hip fracture were allocated to two groups receiving the two forms of Ca without placebo. FS BMD changes in Ca-supplemented non-fractured women were significantly different from those in the placebo group (+0.6 +/- 0.5% v -1.2 +/- 0.7%, p < 0.05). There was no difference in effect between the two forms of Ca. The changes of +0.7 +/- 0.8% v -1.7 +/- 1.6% in FN BMD of Ca-supplemented women and the placebo group did not reach statistical significance. In fractured patients, FS, FN and LS BMD changes were -1.3 +/- 0.8, +0.3 +/- 1.6 and +3.1 +/- 1.2% (p < 0.05 for the last). The rate of new vertebral fractures was 74.3 and 106.2 fractures per 1000 patient-years in Ca-supplemented non-fractured subjects and in the placebo group, respectively, and 144.0 in Ca-supplemented fractured patients. Thus, oral Ca supplements prevented a femoral BMD decrease and lowered vertebral fracture rate in the elderly.
Relative contribution of vertebral body and posterior arch in female and male lumbar spine peak bone mass.
Peak bone mass (PBM) is an important reference value in the diagnosis of osteoporosis. It is usually established by determining the areal bone mineral density (BMD in g/cm2) for a given site of the skeleton in young healthy adults. This measurement takes into account both the thickness and the integrated mineral density of the bone scanned. It should therefore be a major determinant of the resistance to mechanical stress. However, in lumbar spine the mean BMD as determined by dual-energy either isotopic or X-ray (DXA) absorptiometry in antero-posterior (ap) view was repeatedly found not to be different between male and female young healthy adults despite the greater volume of lumbar vertebral bodies in males. A greater contribution of the posterior vertebral arch to areal BMD-ap in females than in males could account for such an apparent discrepancy. In order to clarify this issue we have determined in 65 (32 male and 33 female) young healthy adults aged 20-35 years the relative contribution of the vertebral body (VB) and posterior vertebral arch (VA) to BMD and bone mineral content (BMC) of L2-3 measured by both antero-posterior and lateral (lat) scanning using DXA. In young healthy adults mean BMC in antero-posterior view was found not to be significantly different from the total BMC determined by lateral scanning including both VB and VA. This allowed us then to calculate the VA BMC by substracting VB BMC-lat from BMC-ap.(ABSTRACT TRUNCATED AT 250 WORDS)
Peak bone mass.
Peak bone mass, which can be defined as the amount of bony tissue present at the end of the skeletal maturation, is an important determinant of osteoporotic fracture risk. Measurement of bone mass development. The bone mass of a given part of the skeleton is directly dependent upon both its volume or size and the density of the mineralized tissue contained within the periosteal envelope. The techniques of single-1 and dural-energy photon or X-ray absorptiometry measure the so-called 'areal' or 'surface' bone mineral density (BMD), a variable which has been shown to be directly related to bone strength. Bone mass gain during puberty. During puberty the gender difference in bone mass becomes expressed. This difference appears to be essentially due to a more prolonged bone maturation period in males than in females, with a larger increase in bone size and cortical thickness. Puberty affects bone size much more than the volumetric mineral density. There is no significant sex difference in the volumetric trabecular density at the end of pubertal maturation. During puberty, the accumulation rate in areal BMD at both the lumbar spine and femoral neck levels increases to four- to sixfold over a 3- and 4-year period in females and males, respectively. Change in bone mass accumulation rate is less marked in long bone diaphyses. There is an asynchrony between the gain in statural height and bone mass growth. This phenomenon may be responsible for the occurrence of a transient period of a relative increase in bone fragility that may account for the pattern of fracture incidence during adolescence.(ABSTRACT TRUNCATED AT 250 WORDS)
Determination of circulating parathyroid hormone levels and differential diagnosis of hypercalcemia.
Primary hyperparathyroidism is the most frequent cause of hypercalcemia in outpatients. In contrast, this electrolyte disorder is very often associated with cancer when detected in hospital, particularly in the frame of tumors of breast, lung or lympho-hematopoietic system. Hypercalcemia results from an imbalance between the fluxes of calcium entering and leaving the extracellular space. Theses fluxes, mainly those at the levels of bone and kidney, are the main regulators of calcium homeostasis. Depending on the etiology, increases in either bone resorption or renal tubular calcium reabsorption can predominate as the cause of elevated calcemia. Thus, an increment of renal tubular reabsorption of calcium plays a prominent role in hypercalcemia resulting from increased serum concentrations of parathyroid hormone, but can also be detected in 50% of malignant hypercalcemias. The ectopic production of authentic parathyroid hormone has convincingly been demonstrated in very few cases. The syndrome of pseudohyperparathyrodism encountered in malignant hypercalcemia can be accounted for by the tumoral secretion of an analog of parathyroid hormone, parathyroid hormone-related protein. Both proteins, which are produced by different genes located on different chromosomes, interact with the same cell membrane receptors and display identical spectrum of actions. Since they are immunologically quite distinct, there is no cross-reactivity in the various competitive or radiometric immunoassays actually available. The determination of circulating levels of parathyroid hormone is an essential step in the differential diagnosis of hypercalcemias, provided the assays offer adequate sensitivity and specificity. Nowadays, this appears to be generally the case.
Effects of oral calcitriol on bone mineral density in patients with end-stage renal failure.
Evolution of bone mineral density (BMD) at various skeletal sites and the influence of calcitriol on BMD are still poorly documented in patients with terminal renal failure. Using dual photon absorptiometry, we investigated the changes in BMD at the levels of lumbar spine, femoral neck and midfemoral shaft in 21 patients with end-stage renal failure (ESRF) treated with calcitriol (mean dosage +/- SEM: 0.21 +/- 0.02 microgram/day) and compared them to 25 patients with ESRF but not treated with calcitriol (control group) over a period of 20.3 +/- 1.5 and 17.2 +/- 1.2 months, respectively. Lumbar spine BMD increased by 7.7 +/- 3.2%/year in the treated group and decreased by 2.5 +/- 1.3%/year in the control group (P < 0.005). Femoral shaft BMD increased more in treated than in control group (+ 6.7 +/- 2.3 vs. + 1.4 +/- 2.0%/year; P < 0.05) and femoral neck BMD remained stable. PTH levels increased by 92 +/- 121 and 1033 +/- 254 pmol/year (P < 0.01) in the treated group and the controls, respectively. Osteocalcin changes were -2.7 +/- 3.7 and +20.1 +/- 11.7 micrograms/liter (P < 0.05) per year in the same groups. These results indicate that low doses of oral calcitriol in patients with end-stage renal failure were associated with an increase in BMD at the levels of lumbar spine and femoral shaft, and with a stabilization of serum PTH and osteocalcin concentrations.
Transforming growth factor-beta: a down-regulator of the parathyroid hormone-related protein receptor in renal epithelial cells.
We have recently provided evidence for the ability of transforming growth factor-beta 1 (TGF beta) to modulate PTH-related protein (PTHrP)-mediated responses in opossum kidney (OK) cells through reducing the number of PTHrP receptor-binding sites. In the present studies, we investigated the possible mechanisms by which TGF beta might regulate PTHrP receptor density in OK cells, an area that has remained largely unexplored. The steady state level of PTHrP receptor mRNA was time dependently reduced by TGF beta treatment, with the nadir (approximately 3-fold decrease) between 6-10 h, preceding the maximal inhibition on PTHrP receptor binding at 18 h. We then assessed whether the 41% reduction in binding consequent to 18-h TGF beta exposure was reversible. PTHrP-binding activity recovered considerably after 24 h (23% decrease compared with controls) and almost completely by 48 h. However, the addition of monensin or cycloheximide, but not actinomycin (at a dose effective in preventing TGF beta action in this system) during the 24-h recovery period prevented restoration of PTHrP binding. Upon removal of TGF beta, the PTHrP receptor message showed a trend toward recovery in the ensuing 24 h. Therefore, TGF beta provides an example of heterologous desensitization of the PTHrP receptor in OK epithelial cells by decreasing the expression of the receptor message. The desensitization was reversible, and the first 24-h recovery phase was dependent on synthesis and processing of new receptor proteins.
A thymoma as a cause of true ectopic hyperparathyroidism.
Ectopic tumoral secretion of authentic PTH is rare, as only four cases have been convincingly documented by demonstrating the presence of PTH messenger ribonucleic acid in tumor tissue. We report the case of a 25-yr-old male with biochemical alterations typical of primary hyperparathyroidism (elevated calcemia and renal tubular reabsorption of calcium, decreased phosphatemia and maximal tubular reabsorption of phosphate, and increased intact PTH serum levels). Extensive cervical exploration did not reveal any abnormally enlarged parathyroid tissue, but excision of a palpable superior retrosternal mass led to the correction of all abnormal biochemical values. Histological analysis showed a predominantly epithelial thymoma, without any detectable parathyroid gland on serial slices. Tumor extracts contained immunoreactive PTH material, with serial dilutions paralleling PTH standards in an immunoradiometric assay. By contrast, immunoreactive PTH-related protein was absent. Furthermore, on Northern blot analysis, there was a PTH messenger ribonucleic acid transcript with a size similar to that found in parathyroid adenoma or hyperplasia. The thymoma epithelial cells stained positively with antiserum against PTH-(1-34), but negatively with antichromogranin-A antiserum. These results support the ectopic production of authentic PTH by a thymoma and indicate a novel tumoral cause of primary hyperparathyroidism.
Regulation of parathyroid hormone-related protein production in a human lung squamous cell carcinoma line.
The synthesis and release of parathyroid hormone-related protein (PTHrP) could be influenced in a paracrine or autocrine manner by substances present around or inside tumours, such as bone or stromal cell-derived cytokines, factors produced by the tumour itself or by peritumoural inflammatory cells. We investigated the effects of various cytokines known to be synthesized by osteoblasts, stromal cells, leucocytes or cancer cells, on PTHrP production by the human lung squamous cell carcinoma line BEN. The influence of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was studied, and compared with those of insulin-like growth factors-I and -II (IGF-I, IGF-II), or macrophage- or granulocyte-macrophage colony-stimulating factors (M-CSF, GM-CSF). TNF-alpha caused a 1.9 +/- 0.1-fold increase in immunoreactive PTHrP production, which was maximal by 24 h of incubation. IL-6 caused a 2.3 +/- 0.2-fold increase, which was maximal by 16 h. These effects, which were time- and concentration-dependent, were blocked by monoclonal antibodies raised against the corresponding cytokine. An increase of PTHrP mRNA was found in IL-6-treated cells. IGF-I and IGF-II increased PTHrP production by 2.0 +/- 0.3- and 2.3 +/- 0.1-fold respectively. Neither M-CSF nor GM-CSF altered PTHrP production up to 64 h of incubation. PTHrP production was not affected by varying extracellular calcium concentrations, but was decreased by incubation with 100 nmol/l dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)
Parathyroid hormone-related protein: physiology and pathophysiology.
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Stimulation by interleukin-1 of renal calcium reabsorption in thyroparathyroidectomized rats.
Recombinant human interleukin-1 (rhIL-1) can induce an elevation in calcium that has been ascribed exclusively to the stimulation of bone resorption. In the present study, we investigated whether rhIL-1 could also enhance the renal tubular reabsorption of calcium. The chronic influence of recombinant human rhIL-1 on renal calcium transport was investigated in thyroparathyroidectomized rats. Administration of rhIL-1 at the dose of 1.5 micrograms/day sc for 6 days induced a significant elevation in plasma calcium that was associated with a slight but significant decrease in the urinary excretion of calcium. Recording of the urinary calcium excretion expressed per ml glomerular filtrate at various plasma calcium levels, as achieved by acutely infusing calcium gluconate, indicates that rhIL-1 enhanced the tubular reabsorption of calcium. The calculated index of the tubular reabsorption of calcium (TRCal) was significantly increased by rhIL-1 (2.18 +/- 0.14 versus 1.79 +/- 0.07 mmol/l GFR, p < 0.05, in vehicle-treated rats). The change in the renal handling of calcium was not associated with stimulation of the tubular reabsorption of magnesium. Acute administration of a large dose (24 micrograms given in a bolus IV injection) of rhIL-1 enhanced within minutes the urinary excretion of prostaglandin E2. This effect was followed by a significant increase in urinary cAMP excretion and associated with a lower urinary calcium excretion. In conclusion, the results presented in this study indicate that rhIL-1 administered chronically selectively stimulated the tubular reabsorption of calcium. Experimental evidence suggests that this effect is mediated by prostaglandin-induced cAMP generation. These data strongly suggest that changes in the tubular handling of calcium could contribute to rhIL-1-induced hypercalcemia.
Effects of the bisphosphonate tiludronate on bone resorption, calcium balance, and bone mineral density.
Bone resorption inhibitors, such as bisphosphonates, are potentially useful in treatments aimed at increasing bone mass. Among bisphosphonates, tiludronate has proven efficacious in preventing bone loss in postmenopausal women. However, it is not clearly established whether bisphosphonates are more potent when given intermittently or continuously. We investigated the effects of tiludronate on (1) retinoid-stimulated bone resorption in thyroparathyroidectomized rats, (2) calcium balance in intact rats, and (3) bone mineral density (BMD) as measured by dual-energy x-ray absorptiometry at the levels of the lumbar spine, tail, and tibia in 6-month-old rats made osteoporotic by ovariectomy (OVX), in which an intermittent cyclic schedule of treatment was compared to continuous administration. Tiludronate induced a dose-dependent decrease in retinoid-stimulated bone resorption. It increased the intestinal absorption and body retention of calcium. In OVX rats it caused a time- and dose-dependent increase in BMD at the level of the three investigated sites, the effects being maintained for at least 8 weeks after the end of therapy. Continuous and intermittent cyclic regimens appeared to induce similar increases in BMD. These results indicate that tiludronate is efficacious in decreasing bone resorption and increasing calcium balance and bone mineral density in rats.
Parathyroid hormone-related protein and calcium phosphate metabolism.
There is marked homology between the parathyroid hormone (PTH) and PTH-related protein (PTHrP) molecules at the amino terminal but the rest of the molecules are quite different, providing immunologically distinct peptides. However, they interact with the same receptor. Thus, PTHrP mediates biological actions reminiscent of PTH. PTHrP gene is a single copy gene, producing one to three mRNA transcripts through alternative splicing of the carboxy terminal, encoding peptides of 139, 141 or 173 amino acids. Having been recently isolated from malignant tumours, PTHrP is now considered to be the major mediator of humoral hypercalcaemia of malignancy (HHM). The PTH-like effects of PTHrP on the kidney and bone have been well characterized. The increase in renal tubular calcium reabsorption and the reduction in tubular phosphate reabsorption with a concomitant rise in nephrogenous cyclic AMP constitute the pathophysiological changes in the renal handling of calcium and phosphate in HHM. The osteotropic contribution to the malignant hypercalcaemia has been validated by enhanced osteoclastic bone resorption--an indirect effect of the amino terminal portion of the PTHrP molecule on osteoblasts. However, PTHrP has also been detected in a large number of normal adult tissues/organs as well as in human and animal fetuses. Fetal plasma calcium is higher than maternal and this is achieved by active transport of calcium across the placenta. Using ovine placental perfusion models, PTHrP, which is believed to originate from fetal parathyroid glands and the placenta itself, has been demonstrated to sustain this calcium gradient. Active placental transport of magnesium, but not phosphate, was also shown to be enhanced by PTHrP.(ABSTRACT TRUNCATED AT 250 WORDS)