Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes

Abstract

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), and β1-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of β1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.

Keratinocytes Migration Promotion, Proliferation Induction, and Free Radical Injury Prevention by 3-Hydroxytirosol

“…We investigated if HT treatment could also influence the cell migration process. Several signaling events have been described as involved in the regulation of cell migration, extension, and contraction of the cytoskeleton, and in our cell model, HT induced an increase in the migration rate supported by the activation of MMP-9, ERK, PI3 Kinase, Akt, Rac1, Ras, and RhoA important factors for stimulating cell migration and normal tissue remodeling [ 40 , 41 , 42 , 43 , 58 ]. Successful wound healing requires movement of keratinocytes through the extracellular matrix.…”

Platelet-Rich Plasma Promotes the Proliferation of Human Keratinocytes via a Progression of the Cell Cycle. A Role of Prolidase

“…An inverted optical microscope (Nikon, Minato, Tokyo, Japan) with a 40× magnification was employed to capture images of the injury area. The wound closure rate was counted by ImageJ software ( , National Institutes of Health, Bethesda, MD, USA) and calculated as previously described [ 13 ].…”

“…Cells were cultured on a 100 mm dish in FBS-free DMEM with PRP (0.5–5%) for 30 min and 24 h. Then, cells were collected as previously described [ 13 ]. Protein concentration was measured using the Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…After 24 incubation with 5% PRP, cells were treated as previously described [ 13 ]. A confocal laser scanning microscope (BD Pathway 855 Bioimager, Becton Dickinson, Franklin Lakes, NJ, USA) with AttoVision software was used for image visualization.…”

“…PRP can also increase the healing rate of diabetic wounds, reduce the injured area of ulcers, and improve joint function in osteoarthritis [ 5 , 10 , 11 , 12 ]. Recent studies suggest that prolidase could play an important role in the tissue repair process [ 13 ].…”