Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes

Abstract

In the skin epidermis, keratinocytes undergo anchorage-dependent cornification, which gives rise to stratified multilayers, each with a distinct differentiation feature. The active formation of the cornified cell envelope (CCE), an important element in the skin barrier, occurs in keratinocytes of the upper epidermal layers and impacts their terminal differentiation. In the present study, we identified the extracellularly extruded syntaxin-4 as a potent differentiation regulator of epidermal keratinocytes. We found that differentiation stimuli led to the acceleration of syntaxin-4 exposure at the keratinocyte cell surface and that the artificial control of extracellular syntaxin-4, either by the forced expression of several syntaxin-4 mutants with structural alterations at the putative functional core site (AIEPQK), or by using antagonistic circular peptides containing this core sequence, dramatically influenced the CCE formation, with spatial misexpression of TGase1 and involucrin. We also found that the topical application of a peptide that exerted the most prominent antagonistic activity for syntaxin-4, named ST4n1, evidently prevented the formation of the hyperplastic and hyperkeratotic epidermis generated by physical irritation in HR-1 mice skin. Collectively, these results demonstrate that extracellularly extruded syntaxin-4 is a potent regulator of CCE differentiation, and that ST4n1 has potential as a clinically applicable reagent for keratotic skin lesions.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

“…In certain cell types, a subpopulation reportedly extruded extracellular syntaxin-4 to locally propagate signals for morphogenesis and differentiation2730. To determine whether this is also the case in ES cells, extracellular exposure to endogenous syntaxin-4 was investigated in cells grown in LIF-containing medium.…”

“…However, subpopulations of certain plasmalemmal proteins, including syntaxin2 (epimorphin) and syntaxin-4, reportedly translocate across the cell membrane to locally act as non-diffusible signaling molecules262728293031. For example, certain cells of the embryonic carcinoma (EC) cell line F9 extrude syntaxin-4 as membrane-tethered proteins to locally affect the morphology, motility, and cellular context of adjacent cells27.…”

Extracellularly Extruded Syntaxin‐4 Binds to Laminin and Syndecan‐1 to Regulate Mammary Epithelial Morphogenesis

“…The detailed molecular mechanisms for epimorphin in the mammary gland were revealed [Hirai et al, ], whereas those for syntaxin‐4 remain to be elucidated. However, we recently found that syntaxin‐4, as epimorphin in the mammary gland, is also extruded extracellularly in embryonic carcinoma and normal skin keratinocytes; this extracellular subpopulation functions as a morphological differentiation cue in these cell types [Hagiwara et al, ; Kadono et al, ].…”

Membrane‐tethered syntaxin‐4 locally abrogates E‐cadherin function and activates Smad signals, contributing to asymmetric mammary epithelial morphogenesis

“…Immunohistochemistry, Western blotting, and immunocytochemistry, were carried out according to standard protocols using HRP‐, Alexa Fluor 488‐, or Cy3‐labeled secondary antibodies (GE healthcare). To detect cell surface expression of endogenous syntaxin4, non‐permeabilized cells were stained simultaneously for syntaxin4 and a negative control, β‐actin, as described previously . In brief, living cells on chamber slides were incubated with a mixture of affinity‐purified rabbit anti‐syntaxin4 and mouse anti‐β‐actin monoclonal antibodies.…”