The salivary peptide histatin‐1 promotes endothelial cell adhesion, migration, and angiogenesis

Abstract

Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin-1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts However, the participation of histatin-1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin-1 promotes angiogenesis, as shown, using the chick chorioallantoic membrane model, and by an tube formation assay, using both human primary cultured endothelial cells (HUVECs) and the EA.hy926 cell line. Specifically, histatin-1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin-1 increased the recruitment of RIN2, a Rab5-guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin-1-dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin-1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin-1 is a novel proangiogenic factor that may contribute to oral wound healing.-Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina-Berríos, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

Histatins, wound healing, and cell migration

“…As aforementioned, cell‐ECM attachment and spreading are central steps in cell migration, and these are known to require the activation of Rho GTPases (Ridley et al., ). In the latter study, histatin‐1 was shown to activate Rac1 (i.e., increases Rac1‐GTP levels) in endothelial cells via the so‐called “RIN2/Rab5/Rac1 axis” (Torres et al., ; Figure ), a signaling pathway that is relevant for vascular morphogenesis and angiogenesis (Sandri et al., ). Specifically, histatin‐1 promoted the recruitment of RIN2, a Rab5‐GEF within early endosomes, leading to increased Rab5‐GTP levels, and hence, augmented Rac1 activity and endothelial cell spreading.…”

“…However, the precise identity of cognate receptors implicated in histatin‐dependent migration of epithelial cells remains elusive. The scenario becomes more complex, when extending these analyses to other cell types, such as endothelial cells, because the latter have been suggested to require VEGFR during histatin‐induced migration (Torres et al., ; see text below).…”

“…In oral epithelial cells, histatins have been shown to trigger a subset of signaling pathways associated with cell migration, specifically by activating ERK1/2, but not p38MAPK, nor EGFR‐signaling, as shown by pharmacological inhibition experiments in cells exposed to histatin‐2 (Oudhoff et al., ; Oudhoff, Kroeze et al., ). The involvement of ERK1/2 in cell migration was recently extended to endothelial cells treated with histatin‐1 (Torres et al., ). Although studies have pursued the nature of histatin‐cell surface binding and subsequent internalization (Oudhoff et al., ), mechanistic details about their mode of internalization (i.e., clathrin‐dependent or independent pathways) and subsequent endocytic trafficking remain obscure, and it will be relevant to elucidate the underlying signaling pathways triggered, in light of the recent findings suggesting the involvement of the so‐called “signaling endosomes” during histatin‐driven cell migration (Torres et al., ; see section ).…”

“…The involvement of ERK1/2 in cell migration was recently extended to endothelial cells treated with histatin‐1 (Torres et al., ). Although studies have pursued the nature of histatin‐cell surface binding and subsequent internalization (Oudhoff et al., ), mechanistic details about their mode of internalization (i.e., clathrin‐dependent or independent pathways) and subsequent endocytic trafficking remain obscure, and it will be relevant to elucidate the underlying signaling pathways triggered, in light of the recent findings suggesting the involvement of the so‐called “signaling endosomes” during histatin‐driven cell migration (Torres et al., ; see section ).…”

“…In general, the extended effects that histatins, and particularly histatin‐1, have on a plethora of cells lines with different origins are intriguing and include cells ranging from oral keratinocytes, gingival fibroblasts, skin and corneal epithelial cells, as well as dermal fibroblasts. In fact, recent studies have extended the effects of this particular histatin toward another cell type relevant in wound healing, that is, the endothelial cell (Torres et al., ).…”