Cold Atmospheric Plasma Jet Promotes Wound Healing Through CK2-Coordinated PI3K/AKT and MAPK Signaling Pathways

Abstract

The promising role of cold atmospheric plasma jet (CAPJ) treatment in promoting wound healing has been widely documented in therapeutic implications. However, the fact that not all subjects respond equally to CAPJ necessitates the investigation of the underlying cellular mechanisms, which have been rarely understood so far. Given that wound healing is a complex and prolonged process, post plasma-activated medium (PAM) treated keratinocytes were collected at two time points, 2 h (receiving) and 24 h (recovery), for (phospho)proteomic analysis to systematically dissect the molecular basis of CAPJ-promoted wound healing. The receiving (phospho)proteomics datasets, referred to the time point of 2 h, revealed an apparent increase in the phosphorylation of CK2 and its-mediated PI3K/AKT and MAPK signaling pathways, accompanied by a prompted downstream physiological response of cell migration. Additionally, incorporating the network analysis of predicted kinases and their direct interactors, we reiterated that CAPJ influenced cell growth and migration, thereby paving the way for its role in subsequent wound healing processes. Further determining the proteome profiles at recovery phase, which is the time point of 24 h, displayed a totally different view from the receiving proteome which had almost no change. The upregulation of ROBOs/SLITs expression and vesicle trafficking and fusion-related proteins, along with the abundant presence of 14-3-3 family proteins, indicated that the persistent effect of PAM on the wound healing process could potentially promote keratinocyte-fibroblast cross talk and stimulate extracellular matrix synthesis upon epithelialization. Consistent with proteome patterns, CAPJ-treated wound tissues indeed showed a denser and well-organized extracellular matrix architecture, implying hastened epithelialization during wound healing. Collectively, we delineated the molecular basis of CAPJ-accelerated wound healing at early and late responses, providing valuable insights for treatment selection and the development of therapeutic strategies to achieve better outcomes.

Keywords: CK2 kinase; PI3K/AKT and MAPK signaling; cold atmospheric plasma jet (CAPJ); plasma activated medium (PAM); wound healing.

Graphical abstract

Fig. 1

Characterization of proteome and phosphoproteome in 2-h PAM-treated HaCaT cells.A, wound healing assay of HaCaT cells treated for 2 h with or without PAM, following a 24 h incubation. The wound closure ratio (%) is calculated using ImageJ and showed as means ± SD (N = 3). The significance is calculated using two sample t test (∗p-value <0.05). B, workflow of label-free quantitative (phospho)proteomics analysis. C, bar chart shows the identification number of peptide, protein, phosphopeptide, and class I phosphosite from biological triplicate. Gray bars indicate the number from proteome analysis, while white bars are from phosphoproteome analysis. Pie chart showed the distribution of class I phosphosites on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. D, dot plot presents the quantitative reproducibility of proteins (red) and phosphosites (blue) between biological triplicates. The color indicated the value of Pearson’s correlation coefficient. PAM, plasma-activated medium.

Fig. 2

Phosphorylation sites reveal preferential signaling pathways activated by 2 h PAM treatment in HaCaT cells.A, heatmap presents the predicted kinases and their specific substrates in phosphoproteome analysis. The color code of PHOSTRACK score (red-blue) reflected the degree of activation or inhibition of kinase activity, while the color code of PAM/Ctrl ratio displayed the change degree of phosphorylated substrates for each kinase and their distribution over the hypophosphorylated (yellow) and hyperphosphorylated (azure). B, motif analysis of significantly regulated phosphoserine residues (349 sequence windows) using pLogo. X-axis is the positions of the amino acid residues at C terminal or N terminal to the central phosphoresidue (position “0”). Y-axis is the proportional height of the amino acid residues enriched at the specific position in the pool of the queried phosphopeptides. The red horizontal bars on the pLogo correspond to p = 0.05. C, table represents the scoring information of Top 4 enriched motifs analyzed by using PhosphoSitePlus. Z-score indicates the significance of converted p-value (<1e-06). Fold-change (FC) reflects the enrichment of sequence window entities as compared to the background population of motifs. “Foreground matches” refers to the number of matches within the input entities (FG matches), while “background matches” denote the number of matches within the background (BG matches). FC is calculated by dividing (FG matches/FG size) by (BG matches/BG size), where FG size is the number of our input sequences for pSer (366), and BG size is the total number of motifs in database (802,214). PAM, plasma-activated medium.

Fig. 3

Protein network of enriched kinases and proteins with regulated phosphosites in 2 h PAM-treated HaCaT cells.A, all the proteins with significantly regulated phosphorylation events (cyanite nodes, two sample t test, p-value <0.05 without correction) and predicted kinases (white nodes) are submitted to STRING for protein-protein interaction analysis. The interactors with direct interaction and predicted kinases are depicted on the network topology. B, the predicted kinases and their direct interactors in network topology are subjected for KEGG pathway mapping. Bar chart presents the -log10 transformed enrichment significance FDR, which are p-value corrected using Benjamini–Hochberg approach. FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; PAM, plasma-activated medium.

Fig. 4

PAM-induced activation of PI3K/AKT and MAPK signaling in HaCaT cells. Cells are treated with PAM alone or cotreated with PAM and (A and B) ERK inhibitor (SCH772984), or (C and D) CK2 inhibitor (CX4945) for 2 h. Cell lysates are collected for immunoblotting assay against indicated antibodies, accompanying with a control group (Ctrl) treated with normal medium. Bar charts of (B) and (D) represent the densitometry analyses using ImageJ for immunoblots of (A) and (C), respectively, and expressed as phosphoprotein/total protein ratio. Expression level of PI3K is normalized with α-tubulin. E, representative images of wound healing assay in sample groups of Ctrl, PAM, PAM + SCH772984, and PAM + CX4945 for 0 h and 24 h incubation. Yellow dotted lines represent the wound boundary. The scale bar represents 100 μm. F, bar chart illustrated the wound closure ratio (%) of (E). Data were showed as means ± SD of independent triplicate. All significance was calculated using two sample t test. ∗p-value <0.05, ∗∗p-value <0.01. AKT, serine/threonine-protein kinase; CK2, casein kinase 2; MAPK, mitogen-activated protein kinase; PAM, plasma-activated medium; PI3K, phosphoinositide 3-kinase.

Fig. 5

Characterization of late-phase effects in PAM-treated HaCaT cells (recovery proteome).A, workflow of receiving and recovery proteome analyses. Cells are treated with PAM for 2 h (receiving), replaced with normal medium and incubated for additional 24 h (recovery). Protein extracts are subjected to digestion and the peptide sample was analyzed by LC-MS/MS. B, hierarchical clustering (upper panel) revealed two distinct clusters of differentially expressed proteins (DEPs, two sample t test, p-value <0.05). Proteins are clustered on the top, while samples are indicated on left. Color bar shows the Z-score normalized protein abundance. Pathway enrichment is performed using Reactome database and visualized with bubble plot (lower panel). X-axis is the pathway enrichment significance (FDR calculated using Benjamini–Hochberg approach) and Y-axis is pathway coverage (%). C, representative box plot with all points shows the relative abundance of 14-3-3 proteins compared between Ctrl (light color) and PAM (dark color) in receiving (blue box) and recovery (red box) proteomes. Two sample t test without correction was performed for differential significance. Asterisk symbol indicated the significant level (∗p-value <0.05). Y-axis was the protein abundance normalized with Z-score normalization. FDR, false discovery rate; LC-MS/MS, liquid chromatography tandem mass spectrometry; PAM, plasma-activated medium.

Fig. 6

Histological staining of extracellular matrix proteins in CAPJ-treated wound tissues. Rat skin cutaneous wound was treated with CAPJ for 60s. CAPJ-treated wound tissue section (CAPJ), with parallel untreated control (Ctrl), were prepared on day-3, -7, and -14, and subjected for histochemical staining by using Masson’s Trichrome Stain kit for collagen staining (top panel, in dark blue color), Elastic Stain kit for elastin staining (middle panel, in black color), and Alcian Blue Stain kit for hyaluronic acid (HA) staining (button panel, in light-blue color). The red arrows were the extensively stained area for collagen and elastic fibers formation as well as the conjugates of HA and proteoglycans. Right panel correspond to high-magnification images of each cropped area on low-magnification image. Scale bars in low- and high-magnification images are 20 μm and 5 μm, respectively. CAPJ, cold atmospheric plasma jet.

Fig. 7

Brief scheme of CAPJ-modulated molecular mechanisms in wound healing processes. Proteomics analysis revealed that PAM intervention induces signal regulations involved in receptor tyrosine kinase (RTK) signaling via PI3K/AKT and MAPK pathways which promote a myriad of biological responses in wound repair to regulate cell proliferation, migration, and cell-cell communication. In particularly, CK2 functions as a multilevel modulator in the PI3K/AKT and MAPK pathways by directly phosphorylating AKT1 to promote its activity and phosphorylates PTEN to inhibit its phosphatase activity as well as assure the activation of PI3K/AKT signaling. It also phosphorylates ERK activation loop to promote its translocation into the nucleus for transcriptional regulations. These CAPJ-activated phosphosignaling events subsequently influence downstream outcomes related to keratinocyte-fibroblast cross talk and ECM remodeling during epithelization phase of wound closure. AKT, serine/threonine-protein kinase; CAPJ, cold atmospheric plasma jet; CK2, casein kinase 2; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; PAM, plasma-activated medium; PI3K, phosphoinositide 3-kinase.