Nitric oxide activates multiple wound closure–relevant pathways that directly regulate cellular processes such as cell proliferation, migration, differentiation, and apoptosis. Such intracellular proteins regulated by NO include protein kinase C and cGMP-dependent PKG.
32,33 Protein microarray results indicate that although PKC expression was not different among any of the treatment groups (data not shown), we did observe a 633% increase in phosphorylation of PKCα/β2 at threonine 638 in the RMS+MFR treatment compared with RMS alone. Treatments of RMS only and MFR only did not have such an effect. Although phosphorylation of this site is not essential for PKC activity, it serves to prolong activated PKC functionality by inhibiting dephosphorylation of the PKC catalytic domain.
34 Treatments of RMS only also increased cGMP-dependent protein kinase of both PKG1α and PKG1β subunits 48 hours after wounding. Application of MFR alone and after RMS served to further increase the expression of PKG1α/β by approximately 5- and 2-fold, respectively, when compared with RMS only. Nitric oxide–dependent PKG is known to promote the mitogenic response by activating the RAF1/ERK1/2 pathway.
35 However, activation of this pathway appears unlikely, as results indicate increased phosphorylation of the inhibitory domain at RAF1
S259, which prevents RAF1 membrane accumulation and activation.
36 This result is consistent with our data, which indicate decreased ERK1/2 phosphorylation at the activation site T202+Y204/T185+Y187
37,38 (
Table). In addition, PKG has been shown to promote cell survival in response to stress by activating a PI3K/Akt-dependent response.
39,40 Although we did not specifically look at Akt activity, we noted increased phosphorylation of the regulatory subunit PI3K p85/p55
Y467/Y199 in the RMS+MFR group. Phosphorylation of the regulatory subunit promotes assembly and activation of the PI3K signaling complex by removing the inhibitory effects of p85.
41 These data are preliminary but are consistent with our observed findings indicating altered fibroblast sensitivity to NO. Whether strain, NO, or both affect the regulatory subunits of PKC or PI3K remains unknown, but these results offer potential mechanistic approaches to describe the biomechanical roles in strain-induced wound healing.