Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency

Osteopontin (OPN) is a sialic acid-rich, adhesive, extracellular matrix (EC M) protein with Arg-Gly-Asp cell-binding sequence that interacts with sever al integrins, including alpha(v)beta(3), Since the ECM is a key regulator o f mammary gland morphogenesis, and mammary epithelial cells express OPN at elevated levels, we sought to determine whether this protein plays a role i n the postnatal mammary gland development. By generating transgenic mice th at express OPN antisense-RNA (AS-OPN mice) in the mammary epithelia we achi eved suppression of OPN production in this organ. The pregnant AS-OPN mice displayed a lack of mammary alveolar structures, a drastic reduction in the synthesis of beta-casein, whey acidic milk protein, and lactation deficien cy. In agreement with these findings, we uncovered that a mammary cell line , NMuMG, which undergoes both structural and functional differentiation on ECM-coated plates, when transfected with an antisense OPN-cDNA construct, f ailed to undergo such differentiation. Furthermore, the results of gel-inva sion assays demonstrated that these cells manifest elevated matrix metallop roteinase (MMP) activity when OPN expression is significantly reduced. The identity of this proteinase as MMP-2 is confirmed by Western blotting, zymo graphy, and inhibition of its activity by a specific inhibitor, TIMP-2. Tak en together, our results demonstrate, for the first time, an essential role of OPN in mammary gland differentiation and that the molecular mechanism(s ) of its action, at least in part, involves downregulation of MMP-2.