Gino V. Segre

Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein

Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone—related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (−/−) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (−/−) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (−/−) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (−/−) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Prostaglandins as Mediators of Hypercalcemia Associated with Certain Types of Cancer

We investigated the role of prostaglandins in the hypercalcemia associated with neoplasia. In patients with hypercalcemia and solid tumors the excretion of the major urinary metabolite of the E prostaglandins, 7 alpha-hydroxy-5, 11-diketotetranorprostane-1, 16-dioic acid (PGE-M), was significantly greater than normal, P LESS THAN 0.01 (median of 58.4 and 7.1 ng per milligram of creatinine respectively). Slightly elevated values were seen in normocalcemic patients with solid tumors (14.3 ng per milligram). The levels of the metabolite were normal in hypercalcemic patients with either hematologic neoplasia or primary hyperparathyroidism. Immunoreactive parathyroid hormone was undetectable in the plasma of all hypercalcemic patients with solid tumors. Inhibition of prostaglandin synthesis by aspirin or indomethacin reduced excretion of both the urinary metabolite and serum calcium in six hypercalcemic patients with solid tumors and elevated excretion of the metabolite. These findings support the concept that prostaglandins are mediators of the hypercalcemia caused by certain solid tumors.

Calcium Homeostasis in Immobilization: An Example of Resorptive Hypercalciuria

Prolonged immobilization may result in hypercalcemia, hypercalciuria, and osteoporosis. Although bone resorption is central to this syndrome, the mechanism of resorption is uncertain. In particular, the role of systemic calcium-regulating hormones remains unclear. In 14 immobilized subjects we measured fasting calcium excretion, 24-hour urinary calcium excretion during restricted calcium intake, the renal phosphorus threshold, plasma 1,25-dihydroxyvitamin D, nephrogenous cyclic AMP, and immunoreactive parathyroid hormone. Mean serum calcium levels were normal, but fasting and 24-hour calcium excretion were markedly elevated (0.28 mg per deciliter of glomerular filtrate and 314 mg per 24 hours, respectively). The mean levels of serum phosphorus (4.8 mg per deciliter) and the renal phosphorus threshold (4.3 mg per deciliter) were elevated. Mean plasma 1,25-dihydroxyvitamin D was strikingly reduced (9.9 pg per milliliter), as were nephrogenous cyclic (0.64 nmol per deciliter of glomerular filtrate) and immunoreactive parathyroid hormone in both assays. These findings indicate that the parathyroid--1,25-dihydroxyvitamin D axis is suppressed in patients with immobilization-induced hypercalciuria, as would be predicted by a model of resorptive hypercalciuria.

Na + /H + exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na+/H+ exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase Cβ1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2–PTH1R complex markedly activates phospholipase Cβ and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (Gi/o proteins). NHERF-mediated assembly of PTH1R and phospholipase Cβ is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.

Receptors for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related peptide, vasoactive intestinal peptide, glucagonlike peptide 1, growth hormone-releasing hormone, and glucagon belong to a newly discovered G-protein-linked receptor family

Seven receptors with highly homologous structural features have recently been discovered that belong to a new family of seven membrane-spanning receptors within the G-protein-linked receptor superfamily. These seven all bind small peptide ligands, and many have the unique property to activate the G(S) and at least one other G protein.

The Influence of Hypermagnesemia on Serum Calcium and Parathyroid Hormone Levels in Human Subjects

We measured serum concentrations of calcium and parathyroid hormone in seven pregnant women who were receiving intravenous magnesium sulfate for the suppression of premature labor. After administration of magnesium sulfate, the mean (+/- S.E.M.) serum magnesium level rose rapidly from the normal base-line level of 2.0 +/- 0.2 mg per deciliter to 6.1 +/- 0.4 mg per deciliter (0.8 +/- 0.1 to 2.5 +/- 0.2 mmol per liter) (P less than 0.001) at 30 minutes and remained markedly elevated. Concentrations of total and ionized calcium fell gradually in all subjects from normal base-line concentrations, 8.6 +/- 0.2 and 4.4 +/- 0.1 mg per deciliter (2.2 +/- 0.1 and 1.1 +/- 0.03 mmol per liter), respectively, into the hypocalcemic range, reaching a nadir of 7.6 +/- 0.2 and 3.9 +/- 0.1 mg per deciliter (1.9 +/- 0.1 and 0.98 +/- 0.03 mmol per liter), respectively, at three hours (P less than 0.001). Parathyroid hormone levels fell rapidly in response to magnesium infusion, from 13.1 +/- 2.5 to 7.8 +/- 0.7 pg per milliliter at 30 minutes, and were significantly below base-line levels for two hours despite frank hypocalcemia. These results suggest that hypermagnesemia rapidly decreases the secretion of parathyroid hormone in vivo in human subjects and that parathyroid hormone levels remain depressed despite concomitant hypocalcemia. The results also suggest that the hypocalcemia associated with hypermagnesemia may be due in part to the suppressive effects of hypermagnesemia on parathyroid hormone secretion.

Parathyroid Hormone in Human Plasma: IMMUNOCHEMICAL CHARACTERIZATION AND BIOLOGICAL IMPLICATIONS

Antigenic recognition of four anti-bovine parathyroid hormone antisera was characterized by their reactivity with bovine hormonal fragments (1-34, 1-13, 14-34, 19-34, 53-84) and human hormone extracted from parathyroid adenomas. All antisera were found to have antibody populations which recognized more than one antigenic determinant and all antisera differed in their specificity and reactivity for the fragments of bovine hormone. By modification of two antisera, GP-1 and GP-133, by preincubation with excess concentrations of 1-34 or 53-84 fragments, antigenic recognition was restricted to defined regions of the hormonal sequence.When assays using these modified antisera were applied to the study of hormones extracted from glands, greater immunochemical similarities were seen between bovine and human parathyroid hormone using assays that were specific for the measurement of amino-terminal portions of the hormones than of the carboxy-terminal portions.When assays using these antisera were applied to the study of endogenous parathyroid hormone in human plasma, the immunoreactive hormone in the general circulation was shown to substantially lack an amino-terminal portion of the sequence of the intact hormone, including an antigenic determinant requiring all or some of the 14-19 region. This deletion accounts, at least in part, for the immunochemical heterogeneity of plasma parathyroid hormone in man. Radioimmunoassay of fractions of peripheral plasma subjected to gel filtration confirms that the dominant form of the immunoreactive hormone in the general circulation of man is a hormonal fragment that is totally devoid of amino-terminal reactivity. Because of this deletion, it can be concluded that most of the immunoreactive parathyroid hormone in the general circulation of man must be biologically inactive.

Relationship between intact 1–84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity

With the markedly reduced usage of aluminum salts in renal failure, parathyroid hormone (PTH) has become the major determinant of currently seen bone disease. Clinicians now must consider what PTH level should be sought. Too low a level may lead to the aplastic bone lesion (low turnover bone), and too high a level may cause osteitis fibrosa. Furthermore, conventional normal PTH levels may not be a suitable target because of the well-known resistance to PTH in uremic patients. In this report, we derive the PTH levels that best distinguish patients with low and high bone formation states from those with normal bone formation in a group of 175 dialysis patients without aluminum toxicity. Using bone histological parameters, we propose that ideally PTH levels should be maintained between 10 pmol/L (100 pg/mL) and 20 to 30 pmol/L (200 to 300 pg/mL) in chronic dialysis patients, levels two to four times the upper limit of values found in normal subjects.

Metabolism of parathyroid hormone: Physiologic and clinical significance

Abstract Recent findings concerning the metabolism of parathyroid hormone (PTH) and the heterogeneity, or multiple forms, of circulating PTH have spurred widespread investigation of the nature, origin and physiologic significance of the circulating fragments of PTH. Studies of endogenous PTH in man and cow, and of bovine PTH administered to dogs, have been performed utilizing sensitive, specific immunochemical and radiochemical technics. These studies indicate that fragments of the hormone are responsible for most of the immunoreactive PTH (iPTH) detected in the general circulation and that hormonal cleavage occurs after secretion but not in the intravascular space. Studies of parathyroid effluent plasma have established that the principal secretory product of the parathyroid glands is intact hormone; whether other immunoreactive forms of the hormone are also secreted from the gland is uncertain. In all species studied, the predominant form of circulating hormone is the large fragment consisting of the middle and COOH-terminal portions of the molecule; it lacks more than a third of the NH 2 -terminal portion. Because the NH 2 -terminal portion of the hormone sequence is required for biologic activity, this large fragment must be biologically inactive. Analysis of cleavage patterns of intact hormone indicates that an NH 2 -terminal fragment containing the necessary structural requirements for biologic activity may be produced by this cleavage. This may be of considerable physiologic significance regardless of whether this NH 2 -terminal fragment circulates or is present only outside the intravascular space. Although the significance of hormone metabolism is still unclear, the present findings are helpful in the application and interpretation of radioimmunoassays for PTH.