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The mammalian high temperature requirement A (HtrA) proteins are a family of evolutionarily conserved serine proteases, consisting of four homologs (HtrA1-4) that are involved in many cellular processes such as growth, unfolded protein stress response and programmed cell death. In humans, while HtrA1, 2 and 3 are widely expressed in multiple tissues with variable levels, HtrA4 expression is largely restricted to the placenta with the protein released into maternal circulation during pregnancy. This limited expression sets HtrA4 apart from the rest of the family. All four HtrAs are active proteases, and their specific cellular and physiological roles depend on tissue type. The dysregulation of HtrAs has been implicated in many human diseases such as cancer, arthritis, neurogenerative ailments and reproductive disorders. This review first discusses HtrAs broadly and then focuses on the current knowledge of key molecular characteristics of individual human HtrAs, their similarities and differences and their reported physiological functions. HtrAs in other species are also briefly mentioned in the context of understanding the human HtrAs. It then reviews the distinctive involvement of each HtrA in various human diseases, especially cancer and pregnancy complications. It is noteworthy that HtrA4 expression has not yet been reported in any primary tumour samples, suggesting an unlikely involvement of this HtrA in cancer. Collectively, we accentuate that a better understanding of tissue-specific regulation and distinctive physiological and pathological roles of each HtrA will improve our knowledge of many processes that are critical for human health.

Ex vivo studies with first trimester placental tissues are crucial for understanding human placental development, and culturing placental villi in floating conditions is vital to mimic the natural setting. Moreover, being able to cryopreserve these scarce materials for ex vivo studies would open unprecedented avenues, as they are very limited to research and the need to culture freshly adds further hurdles while limiting efficient use. Here we showed that hanging drop method is a simple yet effective approach to culture placental floating villi. We also revealed that these functional units of the early-stage human placenta can be cryopreserved for culturing, and that frozen-thawed villi can regenerate the syncytial layer following denudation. We further illustrated the utility of frozen-thawed tissues to study syncytialization, while validating the importance of HtrA4 in the process which was shown previously in cell models. These represent significant new knowledge to the field of placental biology.

Preeclampsia (PE) is a pregnancy-specific disorder characterized by multi-system involvement, leading to increased perinatal morbidity and mortality as well as long-term cardiovascular damage in both mother and fetus. This study aimed to investigate the alterations and potential role of high temperature requirement factor A4 (Htra4) in early-onset PE. We conducted a comparative analysis of the baseline data between patients with early-onset PE and normal controls, as well as analyzed the correlation of Htra4 levels in the peripheral blood with pregnancy outcomes. Additionally, we investigated the impact of recombinant protein Htra4 on human umbilical vein endothelial cells (HUVECs). Patients with early-onset PE patients exhibited cardiac, hepatic, and renal impairment, as well as elevated levels of Htra4 in peripheral blood but not in umbilical cord blood. Further correlation analysis reveals a significant association between peripheral blood Htra4 levels and adverse pregnancy outcomes in patients with early-onset PE, particularly a strong correlation between maternal blood Htra4 levels and systolic and diastolic blood pressure, suggesting that Htra4 may be associated with impaired vascular function. Histopathological examination revealed inadequate trophoblast cell invasion in the decidua of early-onset PE patients, along with unsuccessful uterine artery remodeling. Additionally, the spiral arterial lumen in the decidua exhibited narrower and irregularly shaped remodeling. Immunofluorescence localization demonstrated the expression of Htra4 in trophoblasts of villous as well as extra-villous lineages. We observed intensified Htra4 staining in the placenta and decidua tissues of patients with early-onset PE. In combination with the pathological alterations of decidual vessels, it is hypothesized that Htra4 might be related to the dysfunction of endothelial cells. Subsequent research indicated that Htra4 induce the onset of oxidative stress, and inflammatory response in HUVECs, suggesting a potential association between the elevated Htra4 levels and vascular endothelial injury in patients with early-onset PE. This study identified a correlation between Htra4 and adverse pregnancy outcomes in patients with early-onset PE, potentially leading to vascular endothelial injury through elevated levels of Htra4.

The expression of High-temperature requirement factor A4 (HtrA4) mRNA is significantly lower in the chorionic villi of patients with recurrent pregnancy loss (RPL) than in the control group. We conducted an investigation into the cellular functions of HtrA4 using the CRISPR/Cas9 system and shRNA-HtrA4 to create knockout BeWo cells and HtrA4 knockdown JEG3 cells. Our results indicated that the knockout BeWo cells exhibited reduced capacity for invasion and fusion, but increased levels of proliferation and migration, with a significantly shortened cell cycle compared to wild-type cells. Wild-type BeWo cells highly expressed cell invasion- and fusion-related factors, while knockout BeWo cells highly expressed migration-, proliferation-, and cell cycle-related factors. The shRNA-HtrA4 JEG3 cells showed a decreased capacity for invasion, but an increased capacity for migration, accompanied by a decrease in the expression of cell invasion-related factors and an increase in migration-related factors. Moreover, our ELISA results revealed that the serum HtrA4 level was lower in patients with RPL than in the controls. These findings suggest that HtrA4 depletion may be associated with placental dysfunction.

The HtrA4 human protease is crucial in placentation and embryo implantation, and its altered level is connected with pre-eclampsia. The meta-analyses of microarray assays revealed that the HtrA4 level is changed in brain tumors and breast and prostate cancers, which suggests its involvement in oncogenesis. In spite of the HtrA4 involvement in important physiological and pathological processes, its function in the cell is poorly understood. In this work, using lung and breast cancer cell lines, we showed for the first time that the full-length HtrA4 and its N-terminally deleted variant promote cancer cell death induced by chemotherapeutic drugs by enhancing apoptosis. The effect is dependent on the HtrA4 proteolytic activity, and the N-terminally deleted HtrA4 is more efficient in the cell death stimulation. Furthermore, HtrA4 increases the effect of chemotherapeutics on the clonogenic potential and motility of cancer cells, and it increases cell cycle arrest at the G2/M phase. HtrA4 may modulate cell death by degrading the anti-apoptotic XIAP protein and also by proteolysis of the executioner pro-caspase 7 and cytoskeletal proteins, actin and β-tubulin. These findings provide new insight into the mechanism of the HtrA4 protease function in cell death and oncogenesis, and they may help to develop new anti-cancer therapeutic strategies.