Eleanor C. Weir

Humoral Hypercalcemia of Cancer

THE syndrome of humoral hypercalcemia of cancer was first described by Fuller Albright in 1941.1 When a patients hypercalcemia and hypophosphatemia resolved after the radiation of a single bone me...

Effects of continuous glucocorticoid infusion on bone metabolism in the rat.

The effects of continuous administration of supraphysiologic doses of dexamethasone (DEX) on bone metabolism were examined in rats. Adult, male, Sprague Dawley rats were infused with DEX at a constant rate of 16.25 μg/day for 19 days. Despite soft tissue catabolism, DEX treatment led to a significant increase in bone volume in all experiments. This was accompanied by a significant gain in femoral weight and calcium content. These findings were also observed in DEX-treated parathyroidectomized animals indicating that intact parathyroid function was not required for this effect. DEX treatment did not affect mean levels of serum calcium or phosphorus but led to significant declines in circulating levels of PTH and 1,25(OH)2D and in the urinary calcium/creatinine ratio. This latter finding was also observed in PTX animals in which 1,25(OH)2D levels did not change. Serum concentrations of osteocalcin and tartrate-resistant acid phosphatase both declined in a time-dependent manner with DEX treatment suggesting a slowing of bone turnover with the net effect favoring formation. However, histomorphometric findings were variable. Two of three experiments demonstrated a decrease in cellular parameters of formation and resorption and in one experiment, these indices increased. Mineralized surface increased with DEX treatment. We conclude that, in marked contrast to the findings in man and certain other species, DEX treatment increases bone mass in rats. This may in part relate to a relatively greater suppression of resorption vis à vis formation.

Synthetic parathyroid hormone-like protein (1–74) is anabolic for bone in vivo

SummaryParathyroid hormone-related protein (PTHRP) has recently been purified from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The gene encoding PTHRP has been cloned, and based on predicted amino acid sequence, polypeptides comprising the first 36 [36Tyr(1–36) PTHRP amide] and 74 [(1–74)PTHRP] amino acids have been synthesized. Human (h) PTHRP (1–36) and (1–74) are potent bone-resorbing agents, and are catabolic for bone in vivo when given continuously at high doses. Bovine parathyroid hormone (bPTH) (1–34) is also catabolic for bone at high dose levels, but when given in low doses for weeks to months, it is anabolic. Although PTHRP possess several PTH-like properties in bone, hPTHRP (1–34) is reported to be only weakly anabolic in vivo. As polypeptide length influences PTHRP action, we evaluated hPTHRP(1–74) as an anabolic agent for bone in vivo. Twenty-four 4-week-old male Sprague-Dawley rats were given daily subcutaneous injections of hPTHRP(1–74) (1 and 2 nmol/100 g body weight, bw), bPTH(1–34) (4 nmol/100 g bw) or vehicle. Rats were sacrificed on day 12, and serum calcium, phosphorus, and 1,25 dihydroxyvitamin D and femoral bone dry weight, calcium content, and hydroxyproline content were measured. Serum calcium and phosphorus were equivalent in all groups. A significant increase in dry bone weight was observed in both PTHRP-treated groups compared with controls. PTHRP also caused a significant, dose-dependent increase in bone calcium and hydroxypro-line content. Results of these studies indicate that PTHRP (1–74) is anabolic for bone in vivo when administered at low-dosage levels for a prolonged period.

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.

Nuclear Factor-κB p50 Is Required for Tumor Necrosis Factor-α-Induced Colony-Stimulating Factor-1 Gene Expression in Osteoblasts1

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-alpha, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-kappaB site in the CSF-1 promoter is required for TNF-alpha-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-kappaB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IkappaB alpha, IkappaB gamma, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-alpha-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-alpha-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-kappaB p50 knock-out mice. Our findings support the conclusion that the NF-kappaB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

Tumor necrosis factor-α induces transcription of the colony-stimulating factor-1 gene in murine osteoblasts

Tumor necrosis factor‐α (TNF‐α) stimulates bone resorption both in vitro and in vivo. The cellular mechanisms for this effect are not known but one pathway may be via release of osteoblast derived factors which stimulate osteoclast formation. Because colony‐stimulating factor‐1 (CSF‐1) is essential for osteoclast progenitor proliferation, we examined the effect of TNF‐α on osteoblast expression of CSF‐1. TNF‐α treatment of MC3T3‐E1 or primary mouse osteoblasts stimulated the secretion of an activity that was mitogenic for a CSF‐1 responsive cell line and was completely neutralized by antiserum to CSF‐1. By Northern analysis, TNF‐α caused a dose and time (3 to 24 h) dependent increase in CSF‐1 transcript expression in MC3T3‐E1 cells. mRNA stability studies using actinomycin D revealed that TNF‐α does not affect CSF‐1 mRNA half‐life in MC3T3‐E1 cells, while nuclear‐run off analysis demonstrated that TNF‐α increases CSF‐1 gene transcription. Cycloheximide treatment of MC3T3‐E1 cells up‐regulated CSF‐1 mRNA, and compared to either agent alone, cycloheximide and TNF‐α in combination resulted in augmentation of CSF‐1 expression. A series of studies using both agonists and inhibitors indicated that TNF‐α‐induced CSF‐1 expression did not involve the arachidonic acid, PKC, or cAMP pathways. These results suggest that TNF‐α induces CSF‐1 expression in osteoblasts by a transcriptional mechanism which is largely independent of new protein synthesis and of the second messenger pathways examined.

Adenylate cyclase-stimulating, bone-resorbing and B TGF-like activities in canine apocrine cell adenocarcinoma of the anal sac.

SummaryCanine apocrine cell adenocarcinoma of the anal sac (APO-AS) is a spontaneously occurring tumor that causes humorally medicated hypercalcemia in 90% of cases. To further define the nature of the responsible mediator in APO-AS, we examined tumor extracts from five APO-AS and four control tumors for adenylate cyclase-stimulating activity (ACSA). All extracts from APO-AS contained potent ACSA, whereas the four control tumors did not. The ACSA extracted from one tumor demonstrated a dose response curve parallel to that of synthetic bovinePTH-(1–34) and was 80% inhibited by Nle8,18,Tyr34 bPTH-(3–34)amide at a concentration of 10−5 M. Extracts from three APO-AS and three control tumors were also examined forin vitro bone-resorbing activity (BRA). All APO-AS contained significant BRA, stimulating resorption 1.47 to 2.13-fold over basal, whereas none of the control tumors stimulated resorption. Purification of one extract using C18 reverse-phase high pressure liquid chromatography (RP-HPLC) resulted in a single sharp peak of ACSA which was 400-fold purified compared with the initial extract. This pool also contained significant bone-resorbing activity, whereas none of the adjacent pools did. Purification of a second extract using sequential CN and C18 RP-HPLC followed by size exclusion HPLC resulted in material that was at least 10,000-fold purified, and showed co-purification of ACSA and B TGF-like activity.