NFκB1/p50 Is Not Required for TNF-stimulated Growth of Primary Mammary Epithelial Cells: Implications for NFκB2/p52 and RelB

NFκB plays an important role in mammary gland development and breast cancer. We previously demonstrated that TNF stimulates growth of mammary epithelial cells (MEC) in a physiologically relevant 3D primary culture system, accompanied by enhanced DNA-binding of the NFκB p50 homodimer. To further understand the mechanism of TNF-stimulated growth of primary MEC, the requirement for NFκB1/p50 and the role of cyclin D1 in TNF-stimulated growth were examined. TNF induced the formation of DNA-binding complexes of p50 and p52 with their coactivator bcl3 in MEC nuclear extracts. Concomitantly, TNF increased the binding of NFκB proteins to the κB site on the cyclin D1 promoter, and increased expression of cyclin D1 mRNA and protein. Using MEC from p50 null mice, we found that p50 was not required for TNF-induced growth nor for up-regulation of cyclin D1. However, TNF induced a p52/RelB NFκB DNA-binding complex in p50 null MEC nuclear extracts. In addition, we found that in wild type MEC, TNF stimulated the occupancy of p52 and RelB on the cyclin D1 promoter κB site, while p50 was present constitutively. These data suggest that in wild type MEC, TNF stimulates the interaction of bcl3 with p50 and p52, and the binding of p52, as well as RelB, to cyclin D1 promoter κB sites, and as a consequence stimulates the growth of MEC. In the absence of p50, p52 and RelB can compensate for p50 in TNF-stimulated growth and cyclin D1 induction in MEC.