Human recombinant transforming growth factor a (TGFa), which binds to the epidermal growth factor (EGF) receptor and causes several biological effects similar to those caused by EGF, was compared with murine EGF for its effects on a number of parameters of bone cell metabolism. TGFa stimulated bone resorption in two organ culture systems, the fetal rat long bone and neonatal mouse calvarial systems. TGFa stimulated bone resorption at concentrations as low as 0.1 ng/ml. TGFa effects on bone resorption in mouse calvariae were inhibited by indomethacin, suggesting that, like EGF, its effects were mediated by prostaglandin synthesis. TGFa had a different time course of action on bone resorption from that of EGF, causing more rapid release of previously incorporated 45Ca from bone cultures, suggesting that TGFa does not function on bone as a simple EGF analogue. TGFa also caused effects on osteoblast function resembling those of EGF. It inhibited alkaline phosphatase activity in cultured rat osteosarcoma cells with the osteoblast phenotype and inhibited collagen synthesis in fetal rat calvaria at concentrations of 1.0 ng/ml. The lowest concentration of TGFa (expressed as nanogram equivalents of EGF per ml) required to produce a response in all of the systems tested was about 1/10th of that needed for EGF to produce a similar effect. These results indicate that TGFa is a potent stimulator of bone resorption and inhibitor of bone formation as assessed by inhibition of collagen synthesis and alkaline phosphatase activity and are consistent with the hypothesis that TGFa may be responsible, at least in part, for the bone resorption associated with some tumors.Transforming growth factor alpha (TGFa) is secreted by a variety of human and rodent tumor cells and binds to the epidermal growth factor (EGF) receptor. TGFa causes many of the known biological effects of EGF (1-3). Multiple peptides of different molecular weights with TGFa activity (as assessed by inhibition of EGF binding and stimulation of anchorage-independent growth) have been detected in culture media and extracts of tumor cells. A small species of 50 amino acids has been purified (4, 5). It is about 40% homologous with EGF (4, 6). Cloning of the cDNA reveals that it is initially synthesized as a larger precursor, which undergoes subsequent posttranslational cleavage. Proper engineering of the sequence coding for the 50 amino acid TGFa has allowed the expression of biologically active human TGFa in Escherichia coli (6).EGF causes osteoclastic bone resorption (7,8) and inhibits bone formation as assessed by collagen synthesis in organ cultures of fetal rat calvariae (7-9) and alkaline phosphatase activity in osteoblast-like cells (10). Since EGF stimulates osteoclastic bone resorption and TGFa competes with EGF for the same receptor, it has been suggested that tumor production of TGFa could be responsible for the bone destruction that is associated with some neoplasms (11,12). Partially purified bone-resorbing activity associated with an animal tumor co...
Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.
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