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Updated: Mar 1, 2026
Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin
Published on: July 14, 2017
The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte
Panjit Chieosilapatham1, François Niyonsaba2, Chanisa Kiatsurayanon1
1Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan; Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Background:
In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown.
Objectives:
To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism.
Methods:
Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit.
Results:
Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP.
Conclusions:
Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes.
Insights
The novel antimicrobial peptide AMP-IBP5 activates skin cells, promoting wound healing. This peptide enhances keratinocyte migration and proliferation via specific cell receptors and signaling pathways.
Area of Science:
- Cutaneous biology and immunology
- Host defense peptides (HDPs)
- Wound healing mechanisms
Background:
- Host defense peptides (HDPs) possess antimicrobial and immunomodulatory properties, influencing keratinocyte functions like cytokine production, proliferation, migration, and wound healing.
- A novel HDP, AMP-IBP5, demonstrates potent antimicrobial activity comparable to established HDPs.
- The immunomodulatory role of AMP-IBP5 in skin remains unexplored.
Purpose of the Study:
- To determine if AMP-IBP5 stimulates keratinocyte activation.
- To elucidate the underlying molecular mechanisms of AMP-IBP5-induced keratinocyte activation.
Main Methods:
- Quantification of cytokine/chemokine and growth factor production using ELISA kits.
- Assessment of keratinocyte migration via in vitro wound closure assays.
- Analysis of cell proliferation using BrdU incorporation and XTT assays.
- Determination of MAPK and NF-κB activation through Western blotting.
- Measurement of intracellular cAMP levels via enzyme immunoassay.
Main Results:
- AMP-IBP5 selectively upregulated the production of IL-8 and VEGF in keratinocytes.
- AMP-IBP5 significantly enhanced keratinocyte migration and proliferation.
- AMP-IBP5-mediated keratinocyte activation involved Mrg X1-X4 receptors, with downstream signaling through MAPK and NF-κB pathways.
- AMP-IBP5 induced activation of MAPK and NF-κB pathways.
- Increased intracellular cAMP levels correlated with AMP-IBP5-induced VEGF production, but not IL-8.
Conclusions:
- AMP-IBP5 exhibits immunomodulatory effects on keratinocytes beyond its antimicrobial activity.
- AMP-IBP5 promotes keratinocyte activation, migration, and proliferation.
- These findings suggest AMP-IBP5 plays a role in the wound healing process by modulating keratinocyte functions.
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