HRas expression is regulated at RNAm level by Ca2+ entry through Ca2+ release activated Ca2+ (CRAC) channels

KARINA FORMOSO; JULIETA MANSILLA RICARTTI ; SEBASTIAN SUSPRREGUY; LUTZ BIRNBAUMER.

Congreso; Reunion Anual de la Sociedad Argentina de Investigación Clínica; 2019

SAIC

In previous results we showed by FRET that Hras protein interacts with Orai1 and also by using Ca2+ imaging with FURA2 method we demonstrated there was a reduction of SOCE in HEK293 cells trasnsfected with HRas, Nras aor Kras. We also showed that dominant positive mutant of Hras (Hras-Q61L) did not reduced SOCE but the dominant negative mutant of Hras (Hras-S17N) did it. All these results confirmed that SOCE was being regulated by the Ras signaling cascade(s).To going on with this model, we tested if SOCE could regulated the Ras signaling casdade. To do that we used MEF wild type and MEF O1KO and amplified by RTPCR the mRNA of cFOS as a marker of Ras activity. Secondly we transfect Hras and/or Orai1 both in HEK293 cells as well as in MEF-wt and MEF-O1KO. Finally we loaded MEF wt and MEF-OIKO with a Ca2+ quelator BAPTA-AM, and analized the impact on Hras and cFOS at mRNA.Our preliminary results showed that Ca2+ entry through Orai1channels (CRAC channels) reduced mRNA of cFOS in MEFwt compared to MEF O1KO cells. On the other hand, the increasment of mRNA of cFOS in MEF O1KO compared to MEF wt due to the transfection of Hras confimed the dependence of cFOS change with Hras system. Secondly, we incubated MEFwt and O1KO cells with BAPTA-AM and by RTPCR we showed a reduction in the mRNA of both Hras a cFOS.Taken toghether our results showed that Ca2+ entry through Orai1channels reduce mRNA and/or activity of hRas and we hipothetized that influx of Ca2+ entry through Orai1channels could be acting like a negative feedback controls the excessive Ca2+ entry by mediates the inactivation of Ras signaling cascade