Understanding the involvement of HtrA4 during syncytiotrophoblast differentiation

The syncytiotrophoblast (STB), the outermost layer of the human placenta, is vital to pregnancy health as it functions as the direct maternal-fetal interface. Being terminally differentiated and multinucleated, STB cannot divide but is formed/sustained by fusion of cytotrophoblasts throughout pregnancy. HtrA4, produced only in placentas of humans and high-order primates, is obligatory for syncytialization, including differentiation of trophoblast stem cells (TSCs) into STB as HtrA4 knockdown (KD) blocks the process; however, how HtrA4 mediates the process remains unknown. The current study aimed to understand how HtrA4 is involved in STB differentiation, by analyzing HtrA4-KD TSCs using mRNA transcriptomics and scanning electron microscopy (SEM). We first investigated how control and HtrA4-KD TSCs, respectively, altered protein-coding gene expressions globally upon stimulation for STB differentiation, then compared the significantly differentially expressed genes (DEGs) to identify the deficiencies of HtrA4-KD cells. Among the DEGs associated with STB differentiation of controls, 25% of them also occurred in HtrA4-KD cells, implying these were HtrA4-independent; in contrast, the remaining 75% DEGs were severely attenuated or completely blocked in HtrA4-KD cells, signifying these were HtrA4-modulated or HtrA4-dependent. Analysis of biological processes, molecular functions, and cellular pathways associated with these distinctive DEGs suggested that early STB differentiation events, such as ceasing cell division and initiating autophagy, were un-influenced by HtrA4. However, many subsequent processes of STB differentiation were partially or fully influenced by HtrA4, some of which included hormone activity, cell surface and extracellular matrix remodeling, adjustments in cell-cell signaling, and restructuring of various components of the cytoplasm/cytoskeleton. Moreover, our data suggested that pathways related to nervous system development were suppressed during STB differentiation, and this was influenced by HtrA4. Furthermore, it was indicated that altering protein binding was a major juncture of HtrA4 influence, and mitigating oxidative stress during STB differentiation might be tightly coupled with HtrA4. By SEM examination of the morphological changes along the time course of STB differentiation, we further visually demonstrated that HtrA4-KD cells commenced the differentiation process, but were blocked from progressing into multinucleated STB. Collectively, these findings provide important new insights into the pivotal role of HtrA4 in STB differentiation.