O cálcio liberado pelo receptor do inositol 1,4,5-trifosfato da isoforma III (ITPR3) ativa a formação de filamentos de actina nu-clear: uma nova via de sinalização com potencial teranóstico para o câncer

Abstract

Calcium (Ca2+) is an important second messenger involved in functions such as proliferation, migration, and regulation of gene expression. This ion is stored in cellular organelles such as the endoplasmic reticulum (ER) and the nuclear envelope. Its release into the cytoplasm begins with the activation of membrane receptors, which in turn activate phospholipase C (PLC). PLCs hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂), generating diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 then binds to IP3 receptors (ITPRs), promoting Ca2+ release into the cytoplasm or nucleus. However, evidence indicates that the cell nucleus possesses its own machinery for Ca2+ release, though the functions of nuclear calcium remain poorly understood. The aim of this study was to investigate the function and interaction of ITPR isoform 3 (ITPR3) with non-muscular myosin IIa (MYH9) and β-actin in the nucleus. Initially, Western blot and mass spectrometry assays were performed on nuclear fractions of U2OS WT and MDCK.2 cells, revealing expression and interaction of β-actin (ACTB) with MYH9 and ITPR3. In parallel, immunofluorescence assays for ITPR3 were conducted in U2OS WT, MDCK, NIH/3T3, MzChA1, and MzChA1 KO cells, where nuclear interaction between MYH9, β-actin, and ITPR3 were observed. Subsequently, Ca2+ assays were performed in U2OS WT and NIH/3T3 cells to evaluate the ability of fetal bovine serum (FBS), ionomycin, and epidermal growth factor (EGF) to stimulate nuclear Ca2+ release. To elucidate the mechanism of nuclear Ca2+ release, U2OS WT and NIH/3T3 cells were transfected with the “Nuclear Actin Chromobody” (nAC), a construct that enables visualization of nuclear actin filaments. These cells were stimulated with the three agonists, and imaging was performed using microscopy. Next, ITPR3 was knockdown in U2OS nAC and NIH/3T3 nAC cells, followed by mycroscopy assays after stimulation with the same agonists. To corroborate these findings, a Ca2+ assay and nuclear actin filament formation assay were conducted in U2OS WT cells infected with adenoviruses expressing only monomeric red fluorescent protein (mRFP), nuclear-localized IP3 buffers (mRFP-IP3-NLS) or nuclear-excluded IP3 (mRFP-IP3-NES), followed by EGF stimulation. Finally, the GEPIA2 database was used to analyze the overexpression of ITPR3, MYH9, and β-actina in various types of tumors. In summary, the study confirms, using multiple techniques, the expression and interaction of ITPR3, MYH9, and β-actina in the nucleus of several cell lines. It also demonstrates that ITPR3 is responsible for nuclear calcium release, which is necessary for the formation of nuclear actin filaments. This signaling pathway may be involved in tumor progression, conferring potential theranostic value to the proteins studied.