Daniela Talarico

Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and α2MRLRP genes

During the process of embryo implantation, trophoblast cells invade deep into uterine stroma and play a key role in establishing fetomaternal exchange of molecules. We have studied the in vivo expression patterns of the molecules of the urokinase system, during the process of mouse embryo implantation and early placentation. The sites of synthesis of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR), plasminogen activator inhibitor type 1 (PAI-1) and alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) transcripts were determined by in situ hybridization. These genes were found to be expressed in a finely regulated pattern. High levels of uPA mRNA were found in invasive trophoblast cells, while the same cells did not appear to synthesize PAI-1. Starting from day 6.5, endothelial cells of newly forming vessels also transcribed uPA gene. uPAR and alpha 2MR/LRP were in all stages expressed by decidual tissue, and their expression domains overlapped in large areas. Immunohistochemistry with uPA and PAI-1 antibodies revealed areas of co-localization of these secreted proteins with the expression domains of uPAR and alpha 2MR/LRP, which is of great interest in view of the role of these two receptors in clearing uPA-PAI-1 complexes. In situ zymography demonstrated the presence of active uPA in the ectoplacental cone region at 7.5 and 8.5 days. Our studies outline the expression of a set of functionally related genes that is well coordinated between fetal and maternal tissues. This coordination may model other physiological and pathological invasive processes.

The urokinase receptor: Structure, regulation and inhibitor-mediated internalization

Abstract The receptor for urokinase plasminogen activator (uPAR) acts as an anchorage site for uPA on the cell surface where it stimulates pro-uPA activation, allows the internalization of uPA:inhibitor and other complexes and sends directly or indirectly signals into the cell that may promote migration, adhesion and growth. It is a GPI-anchored, three-domain protein that belongs to the Ly6 family and is present at the focal and cell-to-cell contacts, where it concentrates uPA activity. Its activity appears to be important to regulate the invasiveness of human cancer cells both in vitro and in vivo, and its inhibition is now a target for antimetastatic therapy.

The PBX-Regulating Protein PREP1 is present in different PBX-complexed forms in mouse

Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

Expression of Matrix Metalloproteinases During Murine Chorioallantoic Placenta Maturation

A large body of experimental evidence supports the participation of two groups of extracellular proteases, matrix metalloproteinases (MMPs), and plasminogen activators/plasmin, in tissue remodeling in physiological and pathological invasion. In the late mouse placenta, several tissue remodeling and cell invasion processes take place. Spongiotrophoblast migration into maternal decidua, as well as decidual extracellular matrix remodeling require the coordinated action of extracellular proteolytic enzymes. Via Northern and in situ hybridization, we have analyzed the spatio‐temporal expression patterns of members of the MMP family (stromelysin‐3, gelatinases A and B), as well as their inhibitors TIMP‐1, ‐2 and ‐3 in late murine placenta (days 10.5 to 18.5 of gestation). Gelatinase activity in placental extracts was assessed by substrate zymography. Gelatinase A and stromelysin‐3 were found to be prominently expressed in decidual tissue; shortly after midpregnancy, the decidual expression patterns of gelatinase A and stromelysin‐3 became overlapping with each other, as well as with the expression domain of TIMP‐2. On the other hand, gelatinase B transcripts were expressed only by trophoblast giant cells at day 10.5, and were downregulated at later stages. TIMP‐1 and TIMP‐3 transcripts were detected in decidual periphery at day 10.5, while later the expression was restricted to the endometrial stroma and spongiotrophoblasts, respectively. The areas of stromelysin‐3 expression were the same (giant trophoblasts) or adjacent (decidua) to those where urokinase (uPA) transcripts were detected, suggesting a possible cooperation between these proteinases in placental remodeling.

Trophoblast giant cells express NF-κB2 during early mouse development

To investigate whether transcription factors of the NF-kappa B family could play a role in early mammalian development, we have analyzed the expression of nfkb1, nfkb2, c-Rel, RelA, RelB, and bcl-3 from 6.5- to 10.5-day mouse embryo implantation sites. Our study shows that nfkb2 mRNA and protein are specifically localized in trophoblast giant cells throughout the stages analyzed. Trophoblast giant cells obtained upon in vitro cultures of 7.5-day ectoplacental cones display NF-kappa B DNA-binding activity that is supershifted by the anti-NF-kappa B2 antibody. Trophoblast giant cells are embryo-derived cells that form an interface between embryonic and maternal tissues during early mouse development; they are involved in decidual remodeling and expansion of the embryonic cavity, placenta formation, and possibly avoidance of maternal immune response to the embryo. Our study suggests that NF-kappa B2 could play a role in the modulation of genes expressed in trophoblast giant cells during the course of early embryogenesis, and therefore be relevant for tissue remodeling and morphogenesis of placenta.

miR-22 suppresses DNA ligase III addiction in multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability. Here we provide evidence that hyper-activation of DNA ligase III (LIG3) is crucial for genomic instability and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Knockdown of LIG3 impairs MM cells viability in vitro and in vivo, suggesting that neoplastic plasmacells are dependent on LIG3-driven repair. To investigate the mechanisms involved in LIG3 expression, we investigated the post-transcriptional regulation. We identified miR-22-3p as effective negative regulator of LIG3 in MM. Enforced expression of miR-22 in MM cells downregulated LIG3 protein, which in turn increased DNA damage inhibiting in vitro and in vivo cell growth. Taken together, our findings demonstrate that myeloma cells are addicted to LIG3, which can be effectively inhibited by miR-22, promoting a novel axis of genome stability regulation.