Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury

Abstract

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/β, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.

Keywords: Cytokine; Phosphoprotein; Phosphorylation; Spatiotemporal distribution; Traumatic injury.

Fig. 1.

Total protein concentration measured across time and location in response to a traumatic injury. Relative fluorescence intensity (RFI) associated with total protein concentrations of the following proteins were assayed across four time points and three different locations following the femur fracture: Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, and p38. Statistically significant differences (p < 0.05) in protein concentration between different locations are marked with matching symbols (* or #). Error bars reflect ± standard error of the mean.

Fig. 2.

Phosphorylation levels measured across time and location in response to a traumatic injury. Levels of phosphorylated protein of Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, and p38 were determined for each time point and location. Concentrations are expressed as relative fluorescence intensity (RFI). Statistically significant differences (p < 0.05) in protein concentration between different locations are marked with matching symbols (* or #). Error bars reflect ± standard error of the mean.

Fig. 3.

Phosphorylation levels measured across time and location in response to a traumatic injury. Levels of phosphorylated protein of GSK-3α/ß, HSP27, IκBα, p70S6K, STAT3 were determined for each time point and location. Concentrations are expressed as relative fluorescence intensity (RFI). Statistically significant differences (p < 0.05) in protein concentration between different locations are marked with matching symbols (* or #). Error bars reflect ± standard error of the mean.

Fig. 4.

Cytokine levels measured across time and location in response to a traumatic injury. Concentrations (ng/g) of IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α were determined for each time point and location. Statistically significant differences (p < 0.05) of cytokine concentration between different locations at each time point are marked with matching symbols (*, #, or %). Error bars reflect ± standard error of the mean. Figure reprinted from Cytokine, 66, H.N. Currie, M.S. Loos, J.A. Vrana, K. Dragan, J.W. Boyd, Spatial cytokine distribution following traumatic injury, 112–118, Copyright 2014, with permission from Elsevier.

Fig. 5.

Canonical scores plot for the identification of the location of injury. Canonical scores for each covariate calculated by quadratic discriminant analysis are plotted. The different locations of injury, at the site (red circles), 1-cm away (green triangles), and on the other uninjured leg (blue squares), were identified. The + signifies the mean of the covariates in each group. The ellipses represents a 95% confidence level and the biplot rays describe the degree of association of a certain covariate with the canonical variables. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

Canonical scores plot for the identification of the time of injury. Canonical scores for each covariate calculated by quadratic discriminant analysis are plotted. The different times of injury, 0 (blue squares), 6 (green triangles), 24 (purple X’s) and 168 (red circles) h were identified. The + signifies the mean of the covariates in each group. The ellipses represents a 95% confidence level and the biplot rays describe the degree of association of a certain covariate with the canonical variables. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)