Receptor of Activated NF-kappaB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.

To investigate the role of Wnt–β-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and 12-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear β-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the β-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3β, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2fx/fx;Bmp4fx/fx mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the β-catenin–BMP2/4–Osx signaling pathway in osteoblasts.

Despite support for receptor of activated NF-κB ligand (RANKL) as a mediator of mammary progesterone action, the extent to which this cytokine can functionally contribute to established progesterone-induced mammary morphogenetic responses in the absence of other presumptive effectors is still unclear. To address this uncertainty, we developed an innovative bigenic system for the doxycycline-inducible expression of RANKL in the mammary epithelium of the progesterone receptor knockout (PRKO) mouse. In response to acute doxycycline exposure, RANKL is specifically expressed in the estrogen receptor α (ER) positive/progesterone receptor negative (ER(+)/PR(-)) cell type in the PRKO mammary epithelium, a cell type that is equivalent to the ER(+)/PR(+) cell type in the wild-type (WT) mammary epithelium. Notably, the ER(+)/PR(+) mammary cell normally expresses RANKL in the WT mammary epithelium during pregnancy. In this PRKO bigenic system, acute doxycycline-induced expression of RANKL results in ordered mammary ductal side branching and alveologenesis, morphological changes that normally occur in the parous WT mouse. This mammary epithelial expansion is accompanied by significant RANKL-induced luminal epithelial proliferation, which is driven, in part, by indirect induction of cyclin D1. Collectively, our findings support the conclusion that RANKL represents a critical mediator of mammary PR action and that restricted expression of this effector to the ER(+)/PR(+) mammary cell-type is necessary for a spatially ordered morphogenetic response to progesterone.

Four pairs of new polycyclic-meroterpenoid enantiomers, ganocins A-C (1-3) possessing a spiro[4,5]decane ring system, along with ganocin D (4) with an eight-membered ring, were isolated from the fruiting bodies of Ganoderma cochlear. Their structures were determined by spectroscopic data and X-ray diffraction crystallography. Their anti-AChE activities were evaluated, and a possible biogenetic pathway was also proposed.

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP-2 acts synergistically with β-catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross-talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3-E1 cells to investigate the effect of BMP-2 on β-catenin signaling. We found that BMP-2 stimulates Lrp5 expression and inhibits the expression of β-TrCP, the F-box E3 ligase responsible for β-catenin degradation and subsequently increases β-catenin protein levels in osteoblasts. In vitro deletion of the β-catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP-2 treatment. These findings suggest that BMP-2 may regulate osteoblast function in part through modulation of the β-catenin signaling.

SummaryConsidering the regulatory complexities of PR action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR flox ) in mice. Crossing the PR flox/flox mouse with the ZP3-cre transgenic demonstrated that the PR flox allele recombines to a PR null allele (PR d ). Mice homozygous for the recombined null PR allele (PR d/d ) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR Knockout (PRKO) model. Therefore this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage-specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone-responsive target sites.

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To evaluate the possible dietary application of live and heat-inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 AE 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 9 10 8 CFU g À1 live (T1) and heat-inactivated B. pumilus SE5 (T2). The heat-inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat-inactivated probiotic for 60 days (P < 0.05). Furthermore, the heatinactivated probiotic remarkably up-regulated expression of TLR2 and pro-inflammatory cytokines (IL-8 and IL-1b) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat-inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.