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Nisin is a food preservative produced by Lactococcus lactis subsp. lactis. Previous blood biochemical research revealed that nisin has physiological effects in mammals; although the site of action has yet to be identified, keratinocytes have been proposed as a possible target. In this study, we investigated whether nisin affects keratinocytes by examining the effects on eukaryotic intermediate filaments in HaCaT human keratinocytes. Treatment with 93 μg/ml nisin for 24 h decreased the localization of the intermediate filament proteins cytokeratin (CK)5 and CK17 at the cell periphery, which were distributed in a limited area in a ring-or net-like shape. However, this was not observed upon treatment for 6 h. The results of a serial dilution assay revealed that the effect on CK17 localization depends on the nisin concentration and were observed at ≥47 μg/ ml. Moreover, this effect was partially blocked by treatment with the calcium channel blocker bepridil. Thus, despite the long history of nisin as being safe for humans, it has measurable effects on the keratinocyte cytoskeleton. Our findings also indicate that CK5 and CK17 can serve as markers for evaluating the effects of nisin on keratinocytes.

The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP–PKA–ERK–CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca2+ influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.

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