Wound healing (WH) impairment is a well-documented phenomenon in clinical and experimental diabetes. Sex hormones, in addition to a number of signaling pathways including transforming growth factor-β1 (TGF-β1)/Smads and TNF-α/NF-κB in macrophages and fibroblasts, appear to play a cardinal role in determining the rate and nature of WH. We hypothesized that a defect in resolution of inflammation and an enhancement in TNF-α/NF-κB activity induced by estrogen deficiency contribute to the impairment of TGF-βsignaling and delayed WH in diabetes models. Goto-Kakizaki (GK) rats and full thickness excisional wounds were used as models for type 2 diabetes (T2D) and WH, respectively. Parameters related to the various stages of WH were assessed using histomorphometry, western blotting, real-time PCR, immunofluorescence microscopy and ELISA-based assays. Retarded re-epithelialization, suppressed angiogenesis, delayed wound closure, reduced estrogen level and heightened states of oxidative stress were characteristic features of T2D wounds. These abnormalities were associated with a defect in resolution of inflammation, shifts in macrophage phenotypes, increased β3-integrin expression, impaired wound TGF-β1signaling (↓p-Smad2/↑Smad7) and enhanced TNF-α/NFκB activity. Human/rat dermal fibroblasts of T2D, compared to corresponding control values, displayed resistance to TGF-β-mediated responses including cell migration, myofibroblast formation and p-Smad2 generation. A pegylated form of soluble TNF receptor-1 (PEG-sTNF-RI) or estrogen replacement therapy significantly improved re-epithelialization and wound contraction, enhanced TGFβ/Smad signaling, and polarized the differentiation of macrophages toward an M2 or “alternatively” activated phenotype, while limiting secondary inflammatory-mediated injury. Our data suggest that reduced estrogen levels and enhanced TNF-α/NF-κB activity delayed WH in T2D by attenuating TGFβ/Smad signaling and impairing the resolution of inflammation; most of these defects were ameliorated with estrogen and/or PEG-sTNF-RI therapy.

Nrf2 regulates the expression and coordinated induction of a battery of antioxidant phase 2 genes that protect cells against the cumulative damaging effects of oxidative stress (OS), a major contributor in the development of chronic diabetic complications. Using cultured dermal fibroblasts from rats with type 2 diabetes (DFs), we investigated the intracellular redox status and the adaptive response to OS, in which Nrf2 plays a central role. Our data confirmed that the generation of superoxide by NADPH oxidase and the mitochondria was enhanced in DFs compared with corresponding controls. This was associated with a decrease in the antioxidant capacity and an increase in the sensitivity of these DFs to hydrogen peroxide-induced necrotic cell death. Nrf2 levels in total cell extracts were diminished, and this abnormality appears to stem from a diabetes-related decrease in Nrf2 protein stability. Endogenous (oligomycin) and exogenous ( tert-butylhydroquinone) induction of OS enhanced the nuclear translocation of Nrf2 and increased the mRNA expression of Nrf2-sensitive genes in control but not DFs. The activity of the GSK-3β/Fyn axis was increased markedly in DFs when compared with the corresponding controls. Chemical inhibition of GSK-3β mitigated the diabetes-related suppression of the OS-induced nuclear accumulation of Nrf2 and the transcriptional activation of the genes downstream of Nrf2. Overall, these findings suggest that an augmentation in GSK-3β/Fyn signaling during diabetes contributes to a deficit in both the cellular redox state and the Nrf2-based adaptive response to OS. Moreover, they may also offer a new perspective in the understanding and treatment of nonhealing diabetic wounds.

Sarcopenia, a loss of muscle mass and functionality, constitutes a major contributor to disability in diabetes. Hydrogen sulfide (H₂S) dynamics and muscle mass regulatory signaling were studied in GK rats, a model for type 2 diabetes (T2D). GK rats exhibited a number of features that are consistent with sarcopenia and T2D including loss of muscle mass and strength, in addition to glucose intolerance, insulin resistance, and impaired β‐cell responsiveness to glucose. Mechanistically, activation levels of Akt, a key modulator of protein balance, were decreased in T2D. Consequently, we confirmed reduced activity of mTOR signaling components and higher expression of atrophy‐related markers typified by FoxO1/atrogin‐1/MuRF1 and myostatin‐Smad2/3 signaling during the course of diabetes. We observed in GK rat reduced antioxidant capacity (↓GSH/GSSG) and increased expression and activity of NADPH oxidase in connection with augmented rate of oxidation of lipids, proteins, and DNA. H₂S bioavailability and the expression of key enzymes involved in its synthesis were suppressed as a function of diabetes. Interestingly, GK rats receiving NaHS displayed increased muscle Akt/mTOR signaling and decreased expression of myostatin and the FoxO1/MuRF1/atrogin‐dependent pathway. Moreover, diabetes‐induced heightened state of oxidative stress was also ameliorated in response to NaHS therapy. Overall, the current data support the notion that a relationship exists between sarcopenia, heightened state of oxidative stress, and H₂S deficiency at least in the context of diabetes. Moreover, treatment with a potent H₂S donor at an early stage of diabetes is likely to mitigate the development of sarcopenia/frailty and predictably reduces its devastating sequelae of amputation.

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.

Summary: An indolent non-healing wound and insulin and/or insulin-like growth factor (IGF1) resistance are cardinal features of diabetes, inflammation and hypercortisolemia. Little is known about why these phenomena occur in so many contexts. Do the various triggers that induce insulin and/or IGF1 resistance and retard wound healing act through a common mechanism? Cultured dermal fibroblasts from rats and full-thickness excisional wounds were used as models to test the premise that reactive oxygen species (ROS) play a causal role in the development of IGF1 resistance and impaired wound healing under different but pathophysiologically relevant clinical settings, including diabetes, dexamethasone-induced hypercortisolemia and TNFα-induced inflammation. In normal fibroblasts, IGF1 initiated a strong degree of phosphorylation of insulin receptor substrate 1 (IRS1) (Tyr612) and Akt (Ser473), concomitantly with increased PI3K activity. This phenomenon seemed to be attenuated in fibroblasts that had phenotypic features of diabetes, inflammation or hypercortisolemia. Notably, these cells also exhibited an increase in the activity of the ROS−phospho-JNK (p-JNK)−p-IRS1 (Ser307) axis. The above-mentioned defects were reflected functionally by attenuation in IGF1-dependent stimulation of key fibroblast functions, including collagen synthesis and cell proliferation, migration and contraction. The effects of IGF1 on glucose disposal and cutaneous wound healing were also impaired in diabetic or hypercortisolemic rats. The ROS suppressors EUK-134 and α-lipoic acid, or small interfering RNA (siRNA)-mediated silencing of JNK expression, restored IGF1 sensitivity both in vitro and in vivo, and also ameliorated the impairment in IGF1-mediated wound responses during diabetes, inflammation and hypercortisolemia. Our data advance the notion that ROS constitute a convergence nexus for the development of IGF1 resistance and impaired wound healing under different but pathophysiologically relevant clinical settings, with a proof of concept for the beneficial effect of ROS suppressors.