Placental Growth Factor (PlGF) Enhances TLR/MK2 Dependent TNF Gene Transcription and Dissociates miRNAs Targeting TNF Regulatory Transcripts from the Polysome
Authors: Newell Laura F. , Burwick Nicholas , Harrington Christina A , Vartanian Kristina , Burchard Julja , Holtan Shernan G. , Shimamura Akiko , Bagby Grover C. 
Published: October 2019

Abstract
Introduction:
Increasing evidence has demonstrated a cooperative link between angiogenic stimuli and inflammatory responses, though molecular and cellular mechanisms involved in this effect are unclear. Pathologic increases in one such angiogenic factor, placental growth factor (PlGF), a member of the vascular endothelial growth factor family, have been described in diverse clinical settings characterized by an increased risk of inflammatory complications (hemoglobinopathies, inflammatory arthritis, inflammatory bowel disease, and allogeneic hematopoietic cell transplantation). We recently confirmed our hypothesis that the effect of PlGF on the inflammatory response is the result of its capacity to enhance toll-like receptor (TLR)-dependent production of inflammatory cytokines in mononuclear phagocytes (Blood 2015 126:1006). Seeking to identify signaling pathways involved in this cooperative effect, we used TNF gene expression as the paradigm. Of the signaling pathways tested, the most profound effect of PlGF was on the state of activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2). We used loss-of-function approaches, polysome profiling, and microarray to test the hypothesis that PlGF amplifies TLR-dependent TNF gene expression in an MK2-dependent manner and to determine whether the MK2 effect was the result of its known capacity to influence the stability and translation of TNF mRNA.
Methods:
Primary normal donor peripheral blood CD14+ cells were exposed to PlGF (250 ng/ml) and the TLR-7/8 ligand R848 (3 μM), in the presence or absence of small molecule inhibitors of MK2. Secreted TNF protein was measured at 24-hours by ELISA, and mRNA expression at 4-6 hours determined by microarray and qRT-PCR. Actinomycin D chase experiments were performed to determine the effect of PlGF on TNF mRNA decay. RNA from polysome fractions of primary CD14+cells were separated using sucrose density gradient sedimentations, and tested using Affymetrix expression microarrays.
Results:
PlGF alone had no effect on TNF secretion by primary CD14+ cells. However, CD14+ cells exposed to both PlGF and R848 produced significantly more TNF protein than cells exposed to R848 alone (mean 2.35-fold, p=0.0002, n=26). PlGF significantly enhanced R848-induced TNF mRNA expression, compared to R848 alone (mean 2.46-fold, p=0.03, n=12). Immunoblotting demonstrated that: (a) PlGF alone enhanced MK2 phosphorylation compared to untreated cells, and (b) PlGF markedly enhanced R848-induced phosphorylation of MK2 (Figure). While PlGF enhanced total TNF mRNA expression in primary CD14+cells treated with R848, there was no effect of PlGF on TNF mRNA stability in Actinomycin D chase experiments, and no increase in polysome bound TNF mRNA. However, PlGF exposure reduced multiple miRNAs in the polysome fraction including those predicted to target transcription factors known to enhance TNF expression. Microarray confirmed that PlGF increased the abundance of most of those factors, specifically EBF1, EGR1, FOS, NFKB1, and REL. Treatment of primary cells with an MK2 inhibitor (PF-3644022, 1 μM) blocked the effect of PlGF on R848-induced TNF mRNA and protein production.
Conclusions:
PlGF significantly increases TNF production by primary CD14+ cells, and it does so, at least in part, by activating the p38 MAPK/MK2 signaling pathway. Notwithstanding the well-known capacity of MK2 to influence gene expression post-transcriptionally, the mechanism of the PlGF/TLR effect on TNF expression is unambiguously at the point of transcription and independent of TNF mRNA stability or translation, suggesting a novel role for PlGF-dependent MK2 activation in modulating TNF gene transcription. Based on our findings, we hypothesize that PlGF-dependent MK2 activation enhances TNF transcription indirectly by enhancing stability and translation of well-known TNF transcription factors, and this effect involves the PlGF/MK2 dependent disruption of mRNA/miRNA complexes in polysomes, resulting in enhanced stability and translation of the transcription factors EBF1, EGR1, FOS, NFKB1, and REL. These results reveal a previously unrecognized mechanism by which PlGF primes innate immune inflammatory responses to TLR-pathway activation. MK2 inhibitors should be evaluated in preclinical models as a potential treatment option for inflammatory complications in patients with high PlGF states.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.