Characterization of a constitutively active mutant of the human prolactin receptor: From cells to transgenic mice

Knockout studies of prolactin and its cognate receptor (PRLR) have demonstrated an important role for these proteins in mammary gland development suggesting that signaling of the PRLR results in the activation of pathways controlling proliferation, differentiation and suppression of apoptosis. We hypothesized that constitutive activation of the PRLR would activate downstream signaling pathways, resulting in growth factor-independence, transformation in vitro, and increased proliferation/decreased apoptosis in mammary glands in vivo. To test this hypothesis directly, I have constructed a constitutively active mutant of the hPRLR by deleting 178 amino acids in the extracellular ligand-binding domain. Expression of the Δ178 deletion mutant induced growth factor-independent proliferation and constitutive activation of signaling molecules in growth factor-dependent murine myeloid 32Dcl3 and the Nb2 T-cell lymphoma cell lines. In addition, expression of the Δ178 deletion mutant suppressed the apoptosis that occurs when 32Dcl3 cells are cultured in the absence of interleukin-3. Expression of the Δ178 hPRLR deletion mutant in mouse mammary epithelial cell line HC11 and human mammary epithelial cell line MCF12A resulted in the constitutive activation of signaling molecules, and induction of growth factor-independent proliferation of these cells indicating that the PRLR can induce proliferation of mammary epithelial cells. Transgenic mouse lines were generated in which expression of the constitutively active Δ178 hPRLR deletion mutant is targeted to the mammary gland through the use of the mouse mammary tumor virus (MMTV) promoter. These transgenic mice show precocious development in the mammary gland in early virgin and early pregnancy stages, when compared to the control FVB mice. Increased numbers of terminal end buds (TEBs) and terminal ducts (TDs) were observed in transgenic virgin mice and a greater degree of lobuloalveolar formation and ductal branching was observed in early pregnancy. Injections of virgin mice with estrogen plus progesterone indicated that these effects were directly due to the transgene expression and not the hormonal effects of the estrous cycle. Mammary gland involution was also delayed in transgenic mice. There was an increase in the number of mammary epithelial cells and alveoli in transgenic mice 4–6 days post forced weaning compared to the control FVB mice. These studies provide additional evidence that the PRLR plays an important role in mammary gland development and stimulates proliferation of mammary epithelial cells.