The Interaction Between Nodal, Hypoxia-Inducible Factor 1 Alpha, and Thrombospondin 1 Promotes Luteolysis in Equine Corpus Luteum.

Edyta Walewska,Graça Ferreira-Dias,Dariusz Skarzynski,António Galvão,Karolina Wołodko

doi:10.3389/fendo.2019.00667

Abstract

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

Highlights

Molecular regulation of luteolysis is a very intricate process [1]
We first asked if Nodal was able to modulate angiogenic factors expression, and confirmed the level of cluster of differentiation 31 (CD31) protein was significantly downregulated in in vitro culture of mid corpus luteum (CL) explants treated with Nodal (n = 6) (Figure 1A; p < 0.05)
When we treated mid luteal phase CL (mid CL) explants with vascular endothelial growth factor A (VEGFA), we found no changes in Nodal (Figure 1B) and activinlike kinase 4 (Alk4) (Figure 1C), but a trend was visible on activin-like kinase 7 (Alk7) expression inhibition (Figure 1D; p = 0.06)

Summary

Introduction

Molecular regulation of luteolysis is a very intricate process [1]. Following the trigger of the uterine prostaglandin (PG) F2α, various local auto-, paracrine interactions are initiated [1, 2]. The morphogens from transforming growth factor-β (TGF β) superfamily Nodal and TGFβ1 appear to be key for luteolysis in the mare [3]. When we blocked Nodal and TGFβ1 signaling in PGF2α treated cells we abolished its functional and structural luteolytic role [3]. In response to hypoxic conditions, cells develop different strategies such as the transcription of hypoxia-inducible factor 1 (HIF1). HIF1 consists of two subunits: (i) HIF1β, which is constitutively expressed in the nucleus; and (ii) HIF1α, which responds to different factors like cellular O2 tension or other cytokines [7]. HIF1α has been linked to both functional and structural luteolysis [8, 9]

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Conclusion