Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.     

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.