An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

doi:10.1016/j.bbrc.2008.04.103

Biochemical and Biophysical Research Communications

Volume 371, Issue 3, 4 July 2008, Pages 505-509

https://doi.org/10.1016/j.bbrc.2008.04.103 Get rights and content

Abstract

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.

Section snippets

Methods

Cells and reagents. NCI-H292 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA). HUVEC were grown in EGM-2 medium (Cambrex, East Rutherford, NJ). Recombinant human IL-1β was from R&D Systems (Minneapolis, MN). Goat polyclonal anti-RBMX, murine monoclonal anti-TNFR1 (H5), and murine monoclonal anti- β-tubulin (D10) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunoblotting.

RBMX co-immunoprecipitates with ARTS-1

RBMX co-immunoprecipitated with ARTS-1 from membrane proteins isolated from NCI-H292 human pulmonary epithelial cells and was identified by mass spectroscopy (Fig. 1A). Cytokeratin 18, which was pulled-down by both the ARTS-1 antibody and pre-immune serum, was considered to be bound non-specifically. The association between endogenous ARTS-1 and RBMX was confirmed by immunoprecipitation of HUVEC proteins (Fig. 1B).

A subset of RBMX that is not bound by RNA associates with ARTS-1

Since RBMX is a RNA-binding protein, we hypothesized that its association with

Discussion

Here, we sought to identify ARTS-1-associated proteins that participate in cytoplasmic TNFR1 trafficking pathways that mediate the release of TNFR1 to the extracellular compartment. We identified that RBMX co-immunoprecipitated with ARTS-1 from both human airway epithelial and vascular endothelial cells. Using RNA interference, we showed that knock-down of RBMX expression reduced both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1

Acknowledgments

The authors thank Drs. Martha Vaughan, Joel Moss, Christian A. Combs, and Daniella Malide for their helpful discussions and critical review of the manuscript. This research was funded by the Division of Intramural Research, NHLBI, NIH.

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While much of the above data is consistent with RBMX being a tumour suppressor, ectopic expression of RBMY may have an oncogenic role in men in driving liver cancer (Tsuei et al., 2004, 2011; Chua et al., 2015). Although primarily nuclear, RBMX protein physically interacts with a protein called ARTS-1 (aminopeptidase regulator of TNFR1 shedding) to regulate release of tumor necrosis factor receptor (TNFR1) into the extracellular space (Adamik et al., 2008). TNFR1 release is via small extracellular vesicles called exosomes into the circulation.
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¹: Present address: Department of Anesthesiology and Intensive Therapy, Wroclaw Medical University, Wroclaw, Poland.

²: Present address: Division of Pulmonary and Critical Care Medicine, King Hussein Cancer Center, Amman, Jordan.

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An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

Abstract

Section snippets

Methods

RBMX co-immunoprecipitates with ARTS-1

A subset of RBMX that is not bound by RNA associates with ARTS-1

Discussion

Acknowledgments

Cell

J. Biol. Chem.

J. Biol. Chem.

Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors

Proc. Natl. Acad. Sci. USA

Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding

J. Clin. Invest.