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

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-1beta-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1beta-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.

Figure 1. Co-immunoprecipitation of Endogenous RBMX and ARTS-1

Panel A. NCI-H292 cell membrane proteins were immunoprecipitated (IP) with anti-ARTS-1 antibodies or pre-immune serum and stained with Coomassie blue. This gel is representative of two experiments. Panel B. HUVEC lysates were immunoprecipitated with the anti-ARTS-1 antibody or non-immune serum (NI) and RBMX was detected by Western blotting. Panel C. HUVEC lysates, in triplicate, were incubated with RNase inhibitor or a mixture of RNases A and T1 for 1-h prior to immunoprecipitation with the anti-ARTS-1 antibody. RBMX was detected by Western blotting. Panel D. Immunoprecipitation of proteins in HUVEC lysates were performed, in triplicate, as in Panel C. Proteins pulled-down by the ARTS-1 immunoprecipitation are shown in the top panel, while proteins remaining in lysates are shown in the bottom panel labeled supernatant. Panel E. Immunoprecipitation of proteins in HUVEC lysates, performed in triplicate, as described in Panels C and D. TNFR1 was detected by immunoblotting.

Figure 2. RBMX Modulates the Constitutive Release of TNFR1 Exosome-like Vesicles

Panels A and B. TNFR1 in medium from HUVEC transfected with siRNA targeting RBMX or GFP was quantified by ELISA (Panel A) or Western blotting (shown in triplicate in Panel B). Mock denotes cells treated with transfection reagent alone. The asterisk denotes decreased TNFR1 release from cells transfected with siRNA targeting RBMX (P < 0.0001, n = 6). Western blots of HUVEC lysates are also shown. Panel C. Quantification of Western blots from Panel B. The asterisk indicates a significant decrease in the quantity of the 55-kDa TNFR1 present in conditioned medium from cells transfected with siRNA targeting RBMX (P < 0.003, n = 3). Panel D. The quantity of TNFR1 in conditioned medium from HUVEC transfected with plasmids encoding a V5-tagged RBMX (RBMX-V5), empty vector (EV), or the transfection reagent alone (Mock) was detected by ELISA. The asterisk denotes a significant increase in the quantity of TNFR1 present in medium from cells expressing RBMX-V5 (P < 10⁻⁹, n = 6). Panel E. Western blot showing, in triplicate, TNFR1 in HUVEC conditioned medium (top panel) and RBMX-V5 (detected with an anti-V5 antibody), ARTS-1, TNFR1, NUCB2, and β-tubulin in cell lysates (bottom panel). Panel F. Quantification of Western blots from Panel E by densitometry. The asterisk denotes significant increase in the quantity of 55-kDa TNFR1 present in medium from cells transfected with the RBMX-V5 plasmid (P < 10⁻⁴, n = 3).

Figure 3. Down-regulation of RBMX Expression by RNA Interference Attenuates the IL-1β-mediated Proteolytic Cleavage of TNFR1 Ectodomains

Panels A and B. HUVEC transfected with siRNA targeting RBMX or GFP were treated with 20 ng/ml of IL-1β for 2-h and the concentration of TNFR1 in conditioned medium was determined by ELISA (Panel A) or Western blotting (Panel B). Mock denotes treatment with transfection reagent alone. The asterisk indicates that the quantity of TNFR1 in conditioned medium after IL-1β stimulation from cells transfected with siRNA targeting RBMX was significantly reduced (P < 0.0001, n = 6). The blot in Panel B is representative of three individual experiments. Panel C. Quantification of Western blots from Panel B by densitometry. The asterisk indicates a significant decrease in the quantity of the 34-kDa sTNFR1 in conditioned medium after IL-1β stimulation from cells transfected with siRNA targeting RBMX (P < 0.02, n = 3). Panels D – E. HUVEC were transfected with plasmids encoding V5-tagged RBMX (RBMX-V5), the empty vector (EV), or the transfection reagent alone (Mock) for 2 days prior to the addition of fresh medium, without or with 20 ng of IL-1β for 2-h. The quantity of TNFR1 present in conditioned medium was detected by ELISA (Panel D) and Western blotting (Panel E). The asterisk denotes a significant increase in the quantity of TNFR1 present in medium after IL-1β stimulation from cells transfected with the RBMX-V5 plasmid (P < 0.00015, n = 6). This blot in Panel E is representative of three individual experiments. Panel F. Quantification of Western blots from Panel E by densitometry. The asterisk denotes a significant increase in the quantity of 34-kDa TNFR1 present following IL-1β stimulation from cells transfected with the RBMX-V5 plasmid (P < 0.008, n = 3).