Objectives: HtrA4 (high temperature requirement factor A4) belongs to a family of serine proteases that are evolutionarily conserved among all organisms. In general, these proteases play important roles in many cellular processes such as growth, unfolded protein stress response and programmed cell death. In the human, there are four HtrA homologs, whilst HtrA1-3 are widely expressed, HtrA4 expression has so far been detected only in the placenta and its aberrant expression is associated with pregnancy-related diseases. This study aimed to determine whether the expression of HtrA4 is restricted to the placenta of primates and to investigate the potential role of HtrA4 in trophoblast biology.
Methods: Various databases were searched to bioinformatically analyze the expression of HtrA4 in diverse tissues in the human, rhesus monkey and mouse. Comparative analysis was then conducted across these species to determine whether HtrA4 expression is primate specific. The results were then experimentally confirmed by RT-PCR analysis of HtrA4 mRNA in all 3 species, and by immunohistochemical analysis of HtrA4 protein in human and rhesus monkey tissues. Next, the HtrA4 gene was silenced by CRISPR in human trophoblast cell line BeWo and the impact on trophoblast invasion and migration was examined using xCELLIgence.
Results: In both the human and rhesus monkey, HtrA4 mRNA was abundantly expressed only in the placenta, whereas in the mouse, HtrA4 was not expressed in any tissues including the placenta. In the human and rhesus monkey placentas, HtrA4 was highly expressed in the syncytiotrophoblast and extravillous trophoblasts from early stages of pregnancy. HtrA4 silencing in BeWo significantly inhibited cell invasion but did not affect migration.
Conclusions: HtrA4 expression is restricted to the placenta of primates, and it plays an important role in trophoblast invasion which is critical for primate placental development and function.