Fig 1: Loss of ECRG4 increases CD44 expression in vivo.Flow cytometric analysis of blood (A to C) and bone marrow (D to F) from healthy ECRG4 KO mice and littermate controls assessed CD44 expression. (A and D) Representative histograms of CD44 expression on ECRG4 KO (unshaded plot) and WT (shaded plot) neutrophils. (B and E) Middle: Number of CD44+ cells within the CD45+CD11b+Ly6G+ neutrophil population, which were not significantly different between the ECRG4 KO and WT mice (n = 6 mice per group, representative of two independent experiments). (C and F) Right: CD44 MFI on CD45+CD11b+Ly6G+ neutrophils, which show a significant increase in the amount of CD44 expressed per cell in the ECRG4 KO mice compared to their WT controls [**P < 0.01 for (C) and *P < 0.05 for (D); n = 6 mice per group, representative of two independent experiments]. The expression of ECRG4 and CD44 was correlated in vivo in a wound time course. Gene expression was analyzed from an Affymetrix microarray performed on murine cutaneous wounds at multiple times through the wound healing process [Gene Expression Omnibus accession GSE23006; (34)]. Relative expression of CD44 (G) and ECRG4 (H) was plotted against time. (I) To visualize changes in expression of CD44 and ECRG4 together, the data were normalized to their expression at time 0 (uninjured skin). n = 3 mice for each of the eight time points.
Fig 2: Constitutive expression of ECRG4 in HL60 promyelocytes reprograms inflammatory response pathways.RNA-seq was performed on HL60 promyelocytes constitutively expressing ECRG4. (A) The top differentially expressed pathways, as determined by KEGG pathway visualization, included pathways central to inflammatory responses. (B) The top differentially expressed category was proteoglycan signaling, with the CD44 pathway having the most genes altered, as demonstrated in the heat map (N.D., not detected). (C) CD44 cell surface protein was evaluated on these cells via flow cytometry, gating on green fluorescent protein–positive (GFP+) cells and measuring CD44 mean fluorescence intensity (data are representative of five separate experiments with three replicates each; ***P < 0.001). (D) Confirmation of CD44 gene expression in the H60 cells constitutively expressing ECRG4 demonstrated loss of CD44 transcription via quantitative PCR (data are representative of five separate experiments with three replicates each). The ability of a soluble factor from the cells constitutively expressing ECRG4 to suppress CD44 was demonstrated by the addition of conditioned media (CM) to naïve parental HL60 cells. (E) Decreased CD44 cell surface protein expression was determined by flow cytometry (data are representative of five separate experiments with three replicates each). MFI, mean fluorescence intensity.
Fig 3: Model: ECRG4-CD44 dynamics on leukocytes mediate the kinetics of wound inflammation.(A) On quiescent leukocytes, ECRG4 expression maintains CD44 at homeostatic levels. Following injury, leukocytes present in the wound are exposed to proteases during the hemostasis and early inflammatory phases. These proteases cleave leukocyte cell surface ECRG4 to release active peptides into the wound milieu, which enhance the early local inflammatory response. (B) Black tracing represents decreasing cell surface ECRG4 as it is cleaved by wound proteases to release peptides. During this phase, ECRG4 transcription remains low (green tracing), so ECRG4 protein is not replenished on the cell surface. (C) This loss of ECRG4 removes its inhibition of CD44 expression (red tracing), which results in an increase in cell surface CD44 receptor (purple tracing). Increased CD44 functions to resolve the inflammatory phase; it is activated by HA fragments in the wound to decrease proinflammatory signaling. As the wound transitions to the proliferative phase, ECRG4 expression resumes (D) and mediates a decrease in CD44 expression (E), returning both ECRG4 and CD44 cell surface protein to homeostatic levels as wound closure completes and the wound transitions to the remodeling phase. Gene expression data from Fig. 5 (G to I) [Gene Expression Omnibus accession GSE23006; (34)] are shown. Protein expression inferred from published reports (14, 35).
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