Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

BACKGROUND

Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3.

METHODS

The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis.

RESULTS

PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells.

CONCLUSIONS

In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.