HRAS EXPRESSION IS REGULATED AT MRNA LEVEL BY CA2+ ENTRY THROUGH CA2+ RELEASE ACTIVATED CA2+ (CRAC) CHANNELS

KARINA FORMOSO; JULIETA MANSILLA RICARTTI; SEBASTIÁN SUSPERREGUY; LUTZ BIRNBAUMER

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

The store-operated calcium entry (SOCE)channel is the major Ca2+ entry pathway in nonexcitable cells.Store-operated Ca2+ influx is essential for maintaining ER Ca2+content at a precise level and functions in various physiologicalprocesses such as gene transcription, cell-cycle progression, andapoptosis. SOCE is a cellular mechanism linking the calciumdepletion of the endoplasmic reticulum (ER) to the activation ofplasma membrane (PM) Ca2+- permeable channels. The mainscomponents of SOCE are Orai1 and STIM1 proteins. Upon thestimulation of cell surface receptors, like EGFR, depletion of ERCa2+ results in triggering STIM1 translocation to ER-plasmamembrane junctions where they bind and activate Orai1, thepore subunit of the Ca2+ release-activated Ca2+ (CRAC) channel,and resulting in refilling of ER stores. Previously we showed byFRET that Hras protein interacts with Orai1 showed in Ca2+imaging experiments with FURA2 that there was a reduction ofSOCE in HEK293 cells trasnsfected with HRas, Nras or Kras. Wealso showed that a dominant positive mutant of Hras (HrasQ61L) did not reduced SOCE but that the dominant negativemutant of Hras (Hras-S17N) reduced SOCE as did the wt HrasAll these results confirmed that SOCE was being regulated by theRas signaling cascade(s). We next went on to test if SOCE couldregulate the Ras signaling casdade. To this end we used wildtype MEFs and MEFs lacking expression of Orai1 (O1KO MEFs)and amplified by RTPCR the mRNA of cFOS as a marker of Rasactivity. In parallel we transfected Hras and/or Orai1 both inHEK293 cells and in WT and O1KO MEFs. Finally, we loaded MEFwt and MEF-OIKO with the Ca2+ quelator BAPTA-AM, andanalized the impact on Hras and cFOS at mRNAs. Our preliminaryresults showed that Ca2+ entry through Orai1channels (CRACchannels) reduced mRNA of cFOS in WT compared to O1KO MEFscells. On the other hand, the increasment of mRNA of cFOS inO1KO compared to WT MEFs due to the transfection of Hrasconfimed the dependence of cFOS changes on the Hras system.In a final experiment, we incubated WT and O1KO MEFs cellswith BAPTA-AM and by RTPCR we found a reduction in themRNAs of both Hras a cFOS. Taken toghether our results showedthat Ca2+ entry through Orai1channels reduces the mRNAand/or activity of HRas. We hipothetize that influx of Ca2+through Orai1channels may be acting as negative feedbackcontrol on an excessive Ca2+ entry by mediating the inactivationof the Ras signaling cascade