eThe proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1 [IL-1]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg 41 2 (where the down arrow indicates the cleavage location), and potentially by PCs at Arg 41 XXXXArg 46 2. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg 46 2. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIVinduced neuroinflammation, viral infection, and potentially the development of HAND.
BackgroundEstrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear.MethodsMCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation.ResultsIn the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions.ConclusionsOur results suggest that non-genomic signaling by GPER contributes, at least in part, to the survival of breast cancer cells, particularly in the presence of ER-targeted therapies involving SERMs and SERDs. Our results further suggest that GPER expression and FOXO3a localization could be utilized as prognostic markers in breast cancer therapy and that GPER antagonists could promote apoptosis in GPER-positive breast cancers, particularly in combination with chemotherapeutic and ER-targeted drugs, by antagonizing estrogen-mediated FOXO3a inactivation.
In addition to its genomic effects, the steroid hormone estrogen has been shown to mediate rapid signaling events in diverse cell types, including breast cancer cells. These effects are, in part, mediated through its canonical soluble nuclear receptor, ERα. However, a novel estrogen receptor known as GPER (previously termed GPR30) has been demonstrated to activate multiple signaling cascades in response to estrogen. We previously established that PI3Kinase‐mediated production of PIP3 in the nucleus is initiated by estrogen‐mediated stimulation of GPER. The mechanism of this activation and its downstream effects are currently not well understood. Here we describe that rapid signaling through GPER activates PI3Kinase leading to the inactivation and translocation of the proapoptotic protein FOXO3a, resulting in its translocation from the nucleus to the cytoplasm. This activation is mediated by both estrogen and the GPER‐selective agonist G‐1, and occurs via the transactivation of EGFR leading to the activation of the p110alpha subunit of PI3K and subsequent AKT‐mediated phosphorylation of FOXO3a. The nuclear exclusion of FOXO3a by GPER activation suggests GPER signaling results in prosurvival cellular effects in breast cancer cells. Grant Funding Source: NIH
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