Abstract
The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain, and oedema. A role in the modulation or induction of healthy breast tissue growth has been postulated for tissue kallikrein present in human milk. Moreover, tissue kallikrein was found in malignant human breast tissue and bradykinin (BK) stimulates the proliferation of immortalised breast cancer cells. Aim of the present article was to investigate whether BK also exerts mitogenic activity in normal breast epithelial cells and partially characterise the signalling machinery involved. Results show that BK increased up to 2‐fold the 24 h proliferation of breast epithelial cells in primary culture, and that the BK B2 receptor (not B1) inhibitor alone fully blocked the BK response. Intracellular effects of B2 stimulation were the following: (a) the increase of free intracellular Ca^2+^ concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol‐to‐membrane translocation of conventional (PKC)‐α and ‐β isozymes, novel PKC‐δ, ‐ε, and ‐η isozymes; (c) the phosphorylation of the extracellular‐regulated kinase 1 and 2 (ERK1/2); and (d) the stimulation of the expression of c‐Fos protein. EGF, a well known stimulator of cell proliferation, regulated the proliferative response in human epithelial breast cells to the same extent of BK. The effects of BK on proliferation, ERK1/2 phosphorylation, and c‐Fos expression were abolished by GF109203X, which inhibits PKC‐δ isozyme. Conversely, Gö6976, an inhibitor of PKC‐α and ‐β isozymes, and the 18‐h treatment of cells with PMA, that led to the complete down‐regulation of PKC‐α, ‐β, ‐ε, and ‐η, but not of PKC‐δ, did not have any effect, thereby indicating that the PKC‐δ mediates the mitogenic signalling of BK. Phosphoinositide 3‐kinase (PI3K), tyrosine kinase of the epidermal growth factor receptor (EGFR), and mitogen activated protein kinase kinases (MEK) inhibitors were also tested. The results suggest that EGFR, PI3K, and ERK are required for the proliferative effects of BK. In addition, the BK induced cytosol‐to‐membrane translocation of PKC‐δ was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC‐δ. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC‐δ through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation. J. Cell. Physiol. 201: 84–96, 2004. © 2004 Wiley‐Liss, Inc.