Localization, Trafficking and Effects of Histatins on Human Cells

The histatin (Hst) peptide family comprises at least 12 low molecular weight histidine- rich peptides and are found in saliva of higher primates. According to their biological functions, Hsts can be divided into two groups: 1) cell-activating Hsts (e.g. Hst1 and Hst2), and 2) anti-fungal Hsts (e.g. Hst5). Here we focused on the potential mechanisms underlying Hst1’s cell-activating properties. In chapter 2, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in HO1N1 cells Confocal laser scanning microscopy (CLSM) images revealed that F-Hst1, 2 and 5 co-localized with mitotracker-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-tracker-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 cellular metabolic activity. In chapter 3, to further unravel the Hst1 cellular uptake pathway, we investigated the influence of specific inhibitors of GPCR, various endocytosis pathways, ERK signaling, p38 signaling, an inhibitor of mitochondrial respiration, and an inhibitor of Na-K-ATPase activity in the Hst1’s uptake, subcellular targeting, and promoting effect on metabolic activity in epithelial cells. We also adopted live imaging to check the co-localization of Hst1 with early endosomes and late endosomes. We demonstrated that GPCR-endocytosis-ERK signaling regulated the cell-activating effects of human salivary Hst1. In chapter 4, we investigated the stimulatory effect of human salivary Hst1 on the spreading of osteogenic cells in vitro as well as the potential signaling pathways involved. Osteogenic cells were seeded on bio-inert glass slides with or without the presence of Hst1 in dose-dependent or time-course assays. 1 scrambled and 6 truncated Hst1 variants were also evaluated. Cell spreading was analyzed using a well- established point-counting method. Fluorescent microscopy was adopted to examine the cellular uptake of fluorescently labeled Hst1 (F-Hst1) and also the cell spreading on sandblasted and acid-etched titanium surfaces. Signaling inhibitors, such as U0126, SB203580, and pertussis toxin (PTx) were used to identify the potential role of ERK, p38 and GPCR pathways, respectively. Hst1 significantly promoted the spreading of osteogenic cells with an optimal concentration of 10 μM, while truncated and scrambled Hst1 did not. F-Hst1 was taken up and localized in the vicinity of the nuclei. U0126 and SB203580, but not PTx, inhibited the effect of Hst1. 10 μM Hst1 significantly promoted the spreading of osteogenic cells on both bio-inert substrates and titanium SLA surfaces, which involved ERK and p38 signaling. In chapter 5, in this study, we aimed to study the potential relationships between Hst1-targeted mitochondria and cytoskeletal dynamics in HO1N1 cells in vitro. We showed that in the presence of Hst1, an increased mitochondrial mass, increased mitochondrial membrane potential and relative ATP production were found. These observations correlated with increased actin polymerization and filopodia formation. These observations indicate that Hst1 may have a direct action on the cytoskeletal reorganization that precedes cell adhesion. In chapter 6, we discuss our findings in a wider context and provide an overview of Hsts’ cell-stimulating properties, cellular localization and potential biological mechanisms in vitro and in vivo. As discussed in this thesis, research on Hsts has been conducted in various research areas such as wound healing, tissue engineering and angiogenesis. Hst variants have cell-activating functions in vitro studies in various cell lines, such as epithelial cells fibroblasts, endothelial cells, osteoblasts, and chondrocytes. Furthermore, the chapter discusses the possible future studies directions which should be performed to further investigate the involvement of Hst and Hst variants in mechanisms underlying the cell-activating functions which are valuable for a broad spectrum of cell-based and tissue engineering applications.