Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells

Abstract

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control—scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

Human Salivary Histatin-1 Promotes Osteogenic Cell Spreading on Both Bio-Inert Substrates and Titanium SLA Surfaces

“…Hst1 is associated with no cytotoxicity and significantly enhanced metabolic activity of human corneal epithelial cells within 200 µM (Shah et al, 2017). In our recent study, we also show that 10 µM Hst1 significantly promotes metabolic activity of human oral mucosal epithelial cells, skin keratinocytes and gingival fibroblasts (Ma et al, 2020). These findings indicate the good biocompatibility of Hst1.…”

“…Consistent with the previous conclusion (Van Dijk et al, 2017a), we showed that Hst1 significantly enhanced the spreading of osteogenic cells on titanium SLA surfaces when compared to the control group. In our recent study, we showed that Hst1 could be quickly taken up and distribute in the vicinity of surrounding nuclei within epithelial cells and fibroblasts, which suggested an important role of subcellular targets (mitochondria and endoplasmic reticulum) in Hst1's functions (Ma et al, 2020). In this study, our data obtained using fluorescent microscopy showed that F-Hst1 was taken up by osteogenic cells and distributed intracellularly.…”

“…It remains unclear how Hst1 influences cellular metabolic activity. Our recent study shows that Hst1 can be quickly internalized and accumulate in cytoplasm (Ma et al, 2020). In the present study, we adopted CLSM and, for the first time, clearly showed the uptake of Hst1 by preosteoblasts.…”

“…In the present study, we adopted CLSM and, for the first time, clearly showed the uptake of Hst1 by preosteoblasts. After being taken up, Hst1 is quickly targeted to mitochondria and endoplasmic reticulum (ER) (Ma et al, 2020), which suggests a potential role of mitochondria-ER contact in the modulating effects of Hst1 on cellular metabolic activity. However, hitherto, the intracellular signaling pathways and genes activated by Hst1 remains to be elucidated.…”

“…Digital photographs were recorded by a computer integrated in the microscope. To more precisely observe the uptake and localization of F-Hst1, the cells were further studied using a LEICA TCS SP8 confocal laser scanning microscopy (CLSM) system as previously described (Ma et al, 2020). Before incubation with F-Hst1, cell nuclei and membrane were stained with NucBlue TM live cell stain (Life Technologies, Grand Island, NY) and PKH67GL (Sigma-Aldrich, MO, United States) respectively, following manufacturer's instructions.…”