Ectopic expression of the Ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Time for treating bone fracture using rhBMP-2: a randomised placebo controlled mouse fracture trial.

Although the mechanisms of osteoinduction by bone morphogenic proteins (BMPs) are increasingly understood, the most appropriate time to administer BMPs exogenously is yet to be clarified.The purpose of this study was to investigate when BMP may be administered to a fracture arena to maximise the enhancement of healing.Forty mice with externally fixed left femoral fractures were randomised into four groups: Group I, the control group was given a placebo of 30 ll saline at day 0; Groups II, III and IV were given 30 ll saline plus 2.5 lg rhBMP-2, at post-operative days 0, 4 or 8, respectively.Sequential radiographs were taken at days 0, 8, 16.On day 22 the mice were sacrificed and both femora were harvested for biomechanical assessment in 3-point bending and histological evaluation.Radiographic analysis indicated that healing of fractures in Groups II and III was significantly greater (p <0.05) than those in Groups I and IV, at both 16 and 22 days post-fracture. The highest median bone mineral content at the fracture site was evidenced in Group III and II.Furthermore, Group III also had the highest relative ultimate load values, followed by Groups II, IV and I.Greater percentage peak loads were observed between Group I and both Groups II and III (p <0.05). Histological examination confirmed that at 22 days post-fracture, only fractures in Groups II and III had united with woven bone, and Groups I and IV still had considerable amounts of fibrous tissue and cartilage at the fracture gap.Data presented herein indicates that there is a time after fracture when rhBMP administration is most effective, and this may be at the time of surgery as well as in the early fracture healing phases.