Figure 1.
p38 MAPK differentially regulates ERK1/2 and AKT phosphorylation.
70% confluent fibroblasts were pretreated with SB202190 or vehicle DMSO for 1 h and cultured on native collagen or 3DG-collagen for 24 h. Expression of phospho-p38 MAPK (A), and phopsho-ERK1/2 and phospho-AKT, (B) were analyzed by Western blot using whole cell lysates. Total p38 MAPK, total ERK1/2, and total AKT served as loading controls. The bars correspond to the densitometric values of the intensity of both phospho-ERK1/2 bands and the phospho-AKT band compared to that of total ERK1/2 (both bands) and AKT band within each sample, respectively. The density of each sample was then made relative to the density observed in cells cultured on native collagen. All comparisons were made against their respective controls corresponding to native collagen treated with DMSO or 3DG-collagen treated with DMSO. Data are mean ± SD (n = 3), *P<0.01.
Figure 2.
Wound closure rates in fibroblasts pretreated kinase inhibitors.
A. Confluent fibroblasts were pretreated with the inhibitors; p38 MAPK inhibitor SB202190, AKT inhibitor LY294002, or the ERK1/2 inhibitor PD98059 for 1 h and cultured on native collagen or 3DG-collagen. The fibroblasts were then scratched manually with a pipette tip to introduce a wound as previously described. Cell migration into the wound was monitored at 0 h, 24 h, and 48 h by bright field visualization with an epi-fluorescence microscope. For each sample the distance across the wound margin was measured at 10 different points from wound edge to wound edge using Spot software. The measured distance was then converted into a percentage of wound closure when compared to the initial scratch at 0 h as shown in Material and Methods. All statistical comparisons were performed within each time point. Inhibitor comparisons were compared to their respective controls (native collagen or 3DG-collagen). B. To determine that LY294002 or PD98059 did not affect p38 phosphorylation, we pretreated fibroblasts with the AKT or ERK inhibitors for 1 h and cultured the cells on native collagen (COL) or 3DG-collagen (3DG) for 24 h. Whole cell lysates were extracted and phosphorylated p38 MAPK was detected by Western blotting. Phosphorylation of p38 MAPK was normalized to total p38 MAPK. The blot shown is representative of two individual experiments, each producing similar results. All statistical comparisons were performed within each time point. Inhibitor comparisons were compared to their respective controls (native collagen or 3DG-collagen). Data are mean ± SD (n = 3), **P<0.0001, *P<0.001.
Figure 3.
Differential regulation of filopodia extension after fibroblast pretreatment with the p38 MAPK inhibitor SB202190.
Fibroblasts were pretreated for 1 h with the p38 MAPK inhibitor SB202190, or the vehicle DMSO and then cultured on native collagen or 3DG-collagen until confluent. The monolayer of cells was then manually scratched with a pipette tip to introduce the wound. At 4 h post-scratch the fibroblasts were fixed, permeabilized with Triton X-100, and stained with the F-actin dye rhodamine phalloidin. Extension was denoted as filopodia protrusion from initial wound site. The dotted line denotes initial wound site taken at 20 X magnification. Inset picture taken at 40 X magnification. Scale bar represents 10 µm.
Figure 4.
Proliferation rates in fibroblasts pretreated with kinase inhibitors.
4×103 cells/well were pretreated with the p38 MAPK inhibitor SB202190, AKT inhibitor LY294002, or ERK1/2 inhibitor PD98059 for 1 h and then seeded in a 96-well plate containing either native collagen or 3DG-collagen for 0 h, 24 h, and 48 h. At each designated time point the level of proliferation was determined using the cell proliferation reagent WST-1. Quantification of each sample was determined by measuring the absorbance at 450 nm, with a reference wavelength of 600 nm. Comparisons were performed within each time point and compared to their respective controls (native collagen or 3DG-collagen). Data are mean ± SD (n = 3), **P<0.01, *P<0.05.
Figure 5.
Caspase-3 activation after fibroblast pretreatment with kinase inhibitors.
Fibroblasts were pretreated with the inhibitors; p38 MAPK inhibitor SB202190, AKT inhibitor LY294002, or ERK1/2 inhibitor PD98059 for 1 h and then cultured on native collagen or 3DG-collagen for 24 h. Whole cell lysates were assayed for caspase-3 activity at an absorbance of 405 nm. All comparisons were made against their respective controls (native collagen or 3DG-collagen alone). Data are mean ± SD (n = 3), **P<0.0005, *P<0.001.
Figure 6.
Expression of type I collagen after fibroblast pretreatment with kinase inhibitors.
Fibroblasts were pretreated with p38 MAPK inhibitor (SB202190), AKT inhibitor (LY294002), or ERK1/2 inhibitor (PD98059) for 1 h and then cultured on native collagen or 3DG-collagen for 24 h. A, COL1A1 mRNA levels were quantified by real-time RT-PCR and transcripts were normalized to -actin. B, The bar graph was obtained by determining the expression levels of procollagen analyzed by Western blot and normalized to
-actin which was the loading control and the bars correspond to the relative density of procollagen protein compared to that observed in cells cultured on native collagen treated with DMSO. Statistical comparisons were also made against their respective controls (native collagen or 3DG-collagen). Data are mean ± SD (n = 3), *P<0.05.
Figure 7.
Diagram of p38 MAPK regulation of wound healing on native collagen and 3DG-collagen.
Under normal wound healing phosphorylation of p38 MAPK activates AKT and ERK1/2 to promote migration, proliferation, and collagen production. During diabetic wound healing, 3DG-collagen negatively impacts the dermal fibroblast by phosphorylating p38 MAPK to downregulate the expression of ERK1/2 and AKT and promote reduced cell proliferation and migration.