Georgious Theodorou (University Athens) visits University Milan
Effect of various nutrients on expression of plasminogen activator related genes

Professor Antonella Baldi, was the representative of the host University of Milan for the exchange visit of Georgio Theodoroiu (University Athens).

1.Purpose of the visit

The aim of this visit was the assessment of the expression of four plasminogen activator (PA) related genes (urokinase PA, urokinase PA receptor, PA inhibitor type 1 and PA inhibitor type 2) in mammary epithelial cells and adipocytes under various conditions and the analysis of the promoter regions of the same genes by using various plasmid constructs containing different fragments of the promoter regions. The PA system has been well studied in the past and has been strongly linked with lactation and also with milk quality. Further understanding of the system in the molecular level will provide opportunities for manipulation of the system in order to positively affect lactation and milk quality in ruminants. Furthermore the system has been recently shown to be implicated adipogenesis, obesity and the metabolic syndrome and its regulation in adipocytes will provide further understanding of its role in adipose tissue.

2. Description of the work carried out during the visit

BME-UV1 mammary epithelial cells were used in this project, as the particular cell line has been PA assays were performed after treatment with various concentrations of hormones and growth factors thought to have an effect on the PA system. Treatments which exhibited the most pronounced effect were selected for the second part of the experiment.
In the second part of the experiment primer pairs were designed for each of the four genes. The expression of the four genes was assessed using Real Time PCR. According to these results the most significant effects were observed after treatment with dexamethasone, prolactin, epidermal growth factor and insulin growth factor 1. These treatments were subsequently selected for the transient transfection experiments with appropriate reporter gene constructs which included the sequences of the regulatory regions for each of the four PA-related genes that have been previously generated.
The final step of the experimental procedure included the transient transfection of the BME-UV1 cells with the reporter gene constructs. The activity of the reporter constructs was assessed by a luciferase assay and the differences observed between different constructs of the same genes were used in combination with the in silico analysis of the regulatory regions in the functional characterization of the promoters of the PA-related genes.
3T3-L1 adipocytes are at the moment being used following similar experimental protocols to those described above in order to characterize the system and its regulation in the molecular level in the adipose tissue.

3 Description of the main results obtained

The main results obtained from the STSM project point to a differential expression of three out of four PA-related genes (u-PA, u-PAR and PAI-1) in different treatments. The expression of the fourth gene (PAI-2) although detected could not be quantified due to the very low levels of expression in all treatments. Furthermore, regulation of expression for the same three genes was able to be limited in specific regions of the respective promoter sequences depending on the treatment applied.
Although there is a substantial amount of data from the period of the STSM, the project is far from concluded the results obtained show an interesting pattern of expression for the three genes (u-PA, u-PAR and PAI-1) which seems to be regulated by hormones and growth factors that play an important role in lactation. Additional experimental approaches have been planned in order to further investigate the regulation of expression of the PA-related genes in adipocytes and mainly their response to food additives in both mammary epithelial cells and adipocytes.

4 Conclusions

The STSM has been very successful because it has allowed the analysis of the regulation of expression of the PA system in mammary epithelial cells. This system has various physiological roles and also plays a significant role in milk quality and processing. Furthermore, this STSM has allowed that the fellow got trained in the application of several cell culture techniques, which were new for him. This will give him the possibility to apply these techniques in his home institution in future research.

5 Future perspectives

The collaboration with the host institute was considered very successful. Based on the successful collaboration during this short term scientific visit, the host and guest institutes are willing to continue the collaboration. The continuation of this collaboration will include the conclusion of this experimental project by assessing the expression of the PA-related genes and their regulation in an adipocyte cell line (3T3-L1), as well as the effect of the addition of several nutrients to both cell lines.

5 Projected publications/articles resulting or to result from the STSM

The articles to result from this short term scientific mission are currently titled (working titles):
a) “Promoter analysis of ovine urokinase plasminogen activator and its receptor genes by transient transfection”  and
b) “Promoter analysis of ovine plasminogen activator inhibitors type 1 and 2 genes by transient transfection”
These manuscripts are intended to be submitted to the following international journals:
a) “Gene” (impact factor  = 2.416) and/or
b) “Genomics” (impact factor1 = 3.327)
An alternative journal that will be considered is “Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology” (impact factor1 = 1.607). The submission of the articles at the journals is planned by the end of January 2011. The COST action Feed for Health FA0802 will be acknowledged in the resulting publications.

Georgios Theodorou STSM
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