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 6

NF-κB and p-p38 are required for the induction of SNAIL, VCAM1, and IL-6 by HERV-K dUTPase.

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NF-κB and p-p38 are required for the induction of SNAIL, VCAM1, and IL-6...
(A–D) PAECs were transfected with siRNA for p65 or nontargeting siRNA (Con), followed by treatment with 10 μg/mL of HERV-K dUTPase or vehicle as described for Figure 2. (A) SNAIL, IL-6, and VCAM1 mRNA levels, assessed by qPCR. (B) Immunoblot and densitometric quantification of SNAIL protein expression. (C) Secreted IL-6, measured by ELISA in conditioned medium of PAECs following 24-hour treatment with HERV-K dUTPase. (D) Immunoblot and densitometric quantification of VCAM1 protein. (E–H) PAECs were pretreated with 10 μM of p38 inhibitor SB203580 (SB) or with the solvent DMSO (DM) for 2 hours, then treated with HERV-K dUTPase, as described above. (E) Immunoblot and densitometric quantification of p-p65 and p65 protein expression in nuclear extracts, assessed 1 hour after HERV-K dUTPase or vehicle treatment. (F) Immunoblot and densitometric quantification of SNAIL protein expression, assessed after daily HERV-K dUTPase or vehicle treatment as in B. (G) Secreted IL-6, measured by ELISA as in C. (H) Immunoblot and densitometric quantification of VCAM1 protein expression, assessed as described in D. Data are expressed as fold change compared with Veh/Con (A–D) or Veh/DM (E–H) and shown n = 3 with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh/Con (A–D) or Veh/DM (E–H) and #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus H.dUTP/Con or H.dUTP/DM, by a 1-way ANOVA followed by Tukey multiple comparison test.