Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition
Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch
Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch
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Research Article Inflammation Vascular biology

Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition

Abstract

We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response–88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.

Authors

Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch

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Figure 5

MCAM is required for the induction of VCAM1 by HERV-K dUTPase.

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MCAM is required for the induction of VCAM1 by HERV-K dUTPase. (A–D) PAE...
(A–D) PAECs were transfected with siRNA targeting MCAM (MC) or with nontargeting siRNA (Con) for 48 hours, then treated as described for Figure 2. (A) SNAIL, IL-6, and VCAM1 gene expression, assessed in whole cell lysates by qPCR. (B) VCAM1 protein expression was assessed in whole cell lysates by immunoblot and densitometric quantification 72 hours after HERV-K dUTPase treatment in whole cell lysates using GAPDH as a loading control. (C) SNAIL protein in nuclear extracts was assessed by immunoblot and densitometric quantification using Lamin-B as the loading control. (D) Secreted IL-6 was measured by ELISA in the EC media 24 hours following the addition of HERV-K dUTPase. (E–H) PAECs were transfected with siRNAs targeting MCAM and TLR4 (M+T) or with Con before HERV-K dUTPase treatment as described in A–D. (E) MCAM, TLR4, SNAIL, IL-6, and VCAM1 mRNA were assessed as in A. (F) SNAIL protein expression was assessed by immunoblot and densitometric quantification, as in B. (G) Secreted IL-6 was measured by ELISA as in C. (H) VCAM1 protein expression was assessed by immunoblot and densitometric quantification as in D. For all panels, data are expressed as fold change compared with Veh/Con, and show n = 3, mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh/Con and #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus H.dUTP/Con by a 1-way ANOVA followed by Tukey multiple comparison test.