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Oncogenically transnformed mammalian cells irrespective of their origin synthesize and secrete osteopontin (OPN), a sialic acid rich, adhesive, phosphoglycoprotein, not only in excessive amounts but also in different molecular forms, as compared to their non-transformed counterparts. It has been postulated that OPN has important functional roles in oncogenesis, but its mechanism of action remains obscure. In the present study this question was addressed by using Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77 cells) which secrete two discrete molecular forms of OPN, a 69-kDa OPN at the non-permissive temperature (41°C and a 62-kDa form at the permissive temperature (34°C). Investigations were aimed to determine how the two isoforms of OPN secreted by transformed and non-transformed cells originate, whether the two forms have different functional properties, and the effects of specific inhibition of OPN synthesis on the transformed state of the cells. The latter was achieved by transfecting tsB77 cells with antisense OPN cDNA at the permissive temperature. Immunoprecipitation, V8 protease mapping, tryptic peptide analysis, and thrombin digestion confirmed that 62-kDa and 69-kDa proteins are two isoforms of OPN. It was also observed that tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa OPN intracellularly, suggesting that 69-kDa OPN is modified to its 62-kDa form prior to or immediately after secretion by cells at 34ºC. Proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism for the generation of 62-kDa OPN were ruled out, but it was observed that 62-kDa OPN contains significantly reduced levels of sialic acid residues, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface,

The extracellular matrix (ECM) plays an important role in mammary gland development and function. Osteopontin (OPN), a secreted glycophosphoprotein, with several functional and structural properties of ECM proteins, is expressed at elevated levels during normal and pathologic development of the mammary gland and is present in milk. However, whether it plays any developmental role in the mammary gland is unknown. To investigate this possibility, transgenic AS-OPN mice were generated using a transgene expressing OPN antisense RNA under the control of the MMTV-LTR promoter/enhancer. The mammary glands of AS-OPN mice express low levels of OPN, compared to control littermates, show excessive branching of the ductal epithelium at the virgin stage, impaired proliferation of the mammary epithelium, and abnormal alveolar structure formation during pregnancy, and mild to severe lactation deficiency. To further examine the role of OPN at the cellular level, an established murine mammary epithelial cell line (NMuMG), which is capable of differentiation in culture, was transfected with the same DNA construct used to generate AS-OPN mice. Several antisense OPN cDNA transfected clones, which secrete decreased amounts of OPN compared to mock-transfected cells were obtained. These cells were found to have lost their ability to form branched duct-like structures, as judged by branching morphogenesis assays, by growth in collagen gels and stimulation with hepatocyte growth factor. They also failed to spread on type I collagen, although their binding to type IV collagen was unaffected. The antisense transfectants also assumed a mesenchymal phenotype, characterized by fibroblast-like morphology, an apparent loss of cell-cell contacts and spontaneous cell scattering. Transmigration assays and wounding experiments indicated that these cells also have a higher migratory activity than control cells. Northern blot and immunofluorescence analyses showed that migrating cells downregulate OP

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