David D. Thompson

Osteopenia in the immobilized rat hind limb is associated with increased bone resorption and decreased bone formation

The lack of mechanical function (disuse) caused by paralysis, immobilization or weightlessness, leads to osteopenia. This study examines the contribution of bone resorption and bone formation to osteopenia of disuse, during six weeks of limb-immobilization in the growing rat. Male Sprague-Dawley rats of approximately 200 g underwent unilateral hind-limb immobilization by either tenotomy at the knee joint or sciatic neurectomy, while control rats were sham-operated. Animals were sacrificed at 30 and 72 hours, 10, 26 and 42 d postsurgery. Femora were ashed to determine the total mineral content and histomorphometric parameters, static and dynamic, were measured in the secondary spongiosa of the proximal tibial metaphysis. No difference was found in the femoral length of the immobilized legs. Bone loss in the immobilized leg versus the nonimmobilized one, at 10, 26 and 42 d post-surgery was 18.0, 14.0 and 11.2% of femoral mineral content in the tenotomy group, respectively, and 12.4, 16.1 and 15.7% in the neurectomy group. Loss of metaphyseal trabecular bone volume at 10, 26 and 42 days amounted to 67.8, 49.3 and 52.9% in the tenotomy group, respectively, and 70.5, 59.0 and 72.9% in the neurectomy group. The bone loss was caused by: (a) A rapid surge in bone resorption, reflected in a significant increase in the number of osteoclasts per mm bone surface at 30 and 72 h and in the osteoclast surface at 72 h but not at later times, and (b) a sustained decrease in bone mineral apposition rate and bone formation rate (osteoblast-referent) throughout the 42-day immobilization period suggesting osteoblastic hypofunction (reduced activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization-related bone loss in the rat is increased by calcium deficiency.

SummaryThe object of this study was to investigate whether a calcium-deficient diet increases the bone loss produced by mechanical hypofunction (disuse) in the rat. Male Sprague-Dawley rats of approximately 150 g were placed on either a normal diet or a calcium-deficient diet. After 7 days, all rats underwent unilateral hind-limb immobilization by sciatic neurectomy and were sacrificed 30 hours, 72 hours, or 10 days postsurgery. Femora were ashed and the total mineral content (ash weight) was determined. Tibiae were embedded, sectioned, and stained. The metaphyseal secondary spongiosa and the diaphyseal cortical bone were subjected to histomorphometric analysis. Femoral length and serum calcium were not affected by calcium intake or immobilization. Serum parathyroid hormone levels were elevated in rats on the calcium-deficient diet compared to those on the normal diet. Calcium deficiency caused a significant reduction in femoral ash weight (20–35%), tibial cortical thickness (16–20%), and trabecular bone volume (TBV) (33–39%) at 72 hours and 10 days postsurgery. Additional loss of bone mass occurred in the immobilized limb compared to the contralateral intact limb of both dietary groups. This loss occurred earlier (30 hours postsurgery versus 72 hours) in the animals on a calcium-deficient diet and was larger compared to animals on a normal diet (10.6% versus 4.8% at 72 hours and 17.9% versus 12.45% at 10 days). The total bone loss induced by the combination of a calcium-deficient diet and immobilization in this experiment was estimated to equal 46% of femoral ash weight and 79% of tibial TBV. Calcium deficiency increased the number of osteoclasts/mm bone surface in the secondary spongiosa by 25–45%. Immobilization caused a further increase of 50–60% by 72 hours postsurgery. Endosteal mineral apposition rate was significantly decreased (27–44%) by immobilization but was not altered by the low-calcium diet. These data suggest that dietary calcium deficiency augments immobilization-related osteopenia by increasing bone resorption.

A tumor-secreted protein associated with human hypercalcemia of malignancy. Biology and molecular biology.

This investigation addresses a theoretical concept of tumor pathogenesis proposed over 40 years ago, namely that malignancy-associated hypercalcemia can result from endocrine secretion by tumors of a PTH-like factor. These studies demonstrate that a fragment of hHCF alone, without added or tumor-secreted cofactors or hormones, can produce hypercalcemia and other biochemical abnormalities associated with HHM. The hypercalcemia can be generated by hHCF-(1-34)NH2 action on bone, although kidney and gut could contribute to the HHM syndrome when it occurs naturally. No other tumor-secreted peptide displays this biological profile. These studies establish one (PTH-like) mechanism by which human tumors could produce hypercalcemia. Furthermore, the finding that hHCF-(1-34)NH2 is more potent than PTH in some systems is of considerable interest for the future design of hormone analogs. A broad spectrum of biological properties of hHCF-(1-34)NH2, including production of components of the HHM syndrome, can be inhibited by a PTH antagonist. Because [Tyr-34]bPTH-(7-34)NH2 selectively and competitively occupies PTH receptors, our studies demonstrate formally that hHCF-(1-34)NH2 mediates some (and perhaps all) of its actions via receptors conventionally regarded as intended for interaction with PTH, but which actually may be present to allow for expression of bioactivity of both secreted proteins. Although some structural homology is shared by the two hormones and many contribute to interaction with receptors, the disparity in structure, especially within the 1-34 domains responsible for bioactivity in both hormones, is more pronounced. The similarity in biological profiles despite structural differences between hHCF and PTH is emphasized by the inhibitory action of [Tyr-34]bPTH-(7-34)NH2 against the tumor peptide even in the absence of much of the homologous region in the PTH antagonist. This investigation provides impetus for designing more potent antagonists, which must now be regarded more appropriately as inhibitors of both PTH and hHCF. Such antagonists may best be generated from hybrid structures of the two hormones. In any case, these studies establish a promising new approach to therapy of tumor-associated hypercalcemia.

PTH-Like Tumor Hypercalcemia Factor

Hypercalcemia is frequently a serious complication of malignancy. Since in certain patients this condition resembles hyperparathyroidism, a PTH-like factor secreted by the tumor was implicated in this syndrome. Indeed, it was found that several tumors produce a peptide which has PTH-like activity: it stimulates renal and bone adenylate cyclase (Rodan et al., 1983; Stewart et al., 1983), produces bone resorptionin vitro and inhibits phosphate uptake in kidney cells (Strewler et al., 1987). Most recently several tumor-derived peptides were partially sequenced and were found to have sequence similarity to PTH (Moseley et al., 1987; Stewart et al., 1987; Strewart et al., 1987). A complementary DNA sequence cloned from a lung carcinoma cell line (BEN) confirmed the similarity of the deduced N-terminal amino acid sequence to that of PTH, and showed very little similarity to the rest of the molecule (Suva et al., 1987).