Oral Presentation ESA-SRB 2023 in conjunction with ENSA

SPINT1: expression, regulation and function in the developing placenta (#32)

Ciara N Murphy 1 2 , Natalie J Hannan 1 2 , David Simmons 3 , Tuong-Vi Nguyen 1 2 , Ping Cannon 1 2 , Manju Kandel 1 2 , Georgia P Wong 1 2 , Anna Nguyen 1 2 , Natasha Pritchard 2 , Stephen Tong 1 2 , Tu'uhevaha J Kaitu'u-Lino 1 2
  1. Obstetrics, Gynaecology & Newborn Health, The University of Melbourne, Melbourne, Victoria, Australia
  2. Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria, Australia
  3. School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia

Low Serine Peptidase Inhibitor Type I (SPINT1) accompanies fetal growth restriction (FGR) in humans. However, its significance for placental pathophysiology is not yet known. We investigated the expression, regulation, and function of SPINT1 in placentation.

SPINT1 immunohistochemistry and in situ hybridisation on placenta localised protein and mRNA to cytotrophoblast across gestation. Human (cyto)Trophoblast Stem Cells (hTSC) model first trimester placenta. We showed SPINT1 is expressed in hTSCs as well as differentiated lineages; syncytiotrophoblast and extravillous trophoblast (EVT), albeit at lower levels (~50% mRNA, p<0.01) in syncytiotrophoblast.

To explore SPINT1 regulation, protein and mRNA were measured in hTSCs subjected to: (a) Hypoxia (1% O2), relative to first trimester (3%) and term (8%) normoxia. No significant differences were identified comparing 1% vs 3%, however, relative to 8%, hypoxia reduced Spint1 by 40% (p<0.01) and protein secretion by 50% (p<0.01); (b) Knockdown of transcription factors that regulate SPINT1 in other tissues (siRNA; CDX2, GRHL2). This did not alter Spint1 transcripts, although cellular and secreted protein reduced with siGRHL2 (<40%, p<0.01) and nearly doubled with siCDX2 (p<0.01), suggesting post-transcriptional modifications; (c) Inhibiting MMP-mediated secretion using Batimastat, which reduced SPINT1 secretion by 28% (10mM, p<0.05).

SPINT1 was silenced (siRNA) in hTSCs and the effect on cell function assessed. hTSC Proliferation (measured using xCELLigence) decreased after 96 hours (p<0.05).  hTSC differentiation was impaired, with knockdown causing retained cytotrophoblast marker expression TEAD4 (~140% control, p<0.05) in syncytiotrophoblast and 40% reduced EVT marker HLA-G (p<0.05) in EVTs. SPINT1 inhibits a range of proteases, however silencing it had no significant effect on proteolytic activity (fluorogenic substrate).  Although, addition of small molecule SPINT mimetic led to enhanced protease inhibition after 12 hours (reduced 78%, p<0.05).

This work elucidates potential regulators of decreased SPINT1 observed in FGR and some effects this may have on placental development and subsequent function.