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 4

TLR4 and MYD88 mediate the expression of SNAIL and IL-6, but not VCAM1, in response to HERV-K dUTPase.

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TLR4 and MYD88 mediate the expression of SNAIL and IL-6, but not VCAM1, ...
(A–D) PAECs were transfected with siRNA targeting TLR4 (T4) or with nontargeting siRNA (Con) for 48 hours, then treated daily with 10 μg/mL HERV-K dUTPase (H.dUTP) or vehicle (Veh) for 72 hours. (A) SNAIL, IL-6, and VCAM1 gene expression, assessed in whole cell lysates by qPCR. (B) SNAIL protein expression, assessed by immunoblot and densitometric quantification in nuclear extracts, using Lamin-B1 as the loading control. (C) Secreted IL-6, measured by ELISA in the EC media 24 hours following the addition of HERV-K dUTPase. (D) VCAM1 protein expression, assessed by immunoblot and densitometric quantification 72 hours after HERV-K dUTPase treatment in whole cell lysates using GAPDH as a loading control. (E) PAECs were transfected with siRNA targeting MYD88 (MYD) or with Con for 48 hours, then treated daily with 10 μg/mL HERV-K dUTPase (H.dUTP) or vehicle (Veh) for 72 hours. SNAIL, IL-6, and VCAM1 gene expression changes were assessed by qPCR. For all panels, data are expressed as fold change compared with Veh/Con levels, showing the individual data points. n = 3 with 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 < 0.0001 versus H.dUTP/Con by 1-way ANOVA and Tukey multiple comparison test.