SMP30 is expressed in rat mammary gland and down-regulated by estradiol
Cláudio JB Maia, Cecilia RA Santos, Fernando Schmitt & Silvia Socorro
The SMP30 (Senescence Marker Protein 30) is involved in the maintenance of intracellular Ca2+ homeostasis and in the regulation of various Ca2+- dependent proteins. A suppressive effect on cell proliferation, DNA synthesis and on the expression of oncogenes in rat hepatoma cells overexpressing SMP30 has been reported recently suggesting it may have a role in cancer progression. High levels of SMP30 expression have been found in liver and kidney of rats but no studies have focussed so far on the mammary gland where unbalanced calcium homeostasis and signalling is closely associated with its pathophysiology. The goal of the present study was to determine if SMP30 is expressed in rat mammary gland and to study its regulation by 17β-estradiol (E2). For this purpose total RNA was extracted from rat mammary glands, reverse transcribed and subjected to PCR using SMP30 highly specific primers. The identity of the PCR product was confirmed by automatic sequencing. The presence of the SMP30 protein was confirmed by Western blotting of total protein extracts, which showed the presence of the protein as an intense band of ∼30 KDa, and by immunohistochemistry showing that SMP30 localizes preferentially in the citosol. To evaluate the responsiveness of SMP30 to E2, adult females were ovariectomised (n=10) and 5 weeks after surgery they were either implanted with an Alzet mini-osmotic pump delivering 400 μg E2/Kg/day (n=5) or vehicle only (n=5) for 7 days. Sham operated animals (n=5) were also included in the experiment but not implanted. The expression of SMP30 in the mammary gland was analysed by Real-Time PCR and the results showed its downregulation by E2 in the rat mammary gland (P<0.05). These results suggest a likely involvement of SMP30 in breast physiology possibly related to estrogen dependent pathways. Further work to elucidate the SMP30 role in the mammary gland is underway.
Acknowledgements: Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/13388/2003)