Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.     

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process

Several lines of in vitro evidence suggest the potential role of IFN-gamma in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-gamma in the skin wound healing process utilizing WT and IFN-gamma KO mice. In WT mice, excisional wounding induced IFN-gamma mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-gamma KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-gamma KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-gamma KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-beta1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-gamma augmented the TGF-beta1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-gamma/Stat1 and TGF-beta1/Smad signaling pathways in the wound healing process.     

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.     

Novel regulation and dynamics of myosin II activation during epidermal wound responses

Wound healing in the skin is an important and complex process that involves 3-dimensional tissue reorganization, including matrix and chemokine-triggered cell migration, paracrine signaling, and matrix remodeling. The molecular signals and underlying mechanisms that stimulate myosin II activity during skin wound healing have not been elucidated. To begin understanding the signaling pathways involved in the activation of myosin II in this process, we have evaluated myosin II activation in migrating primary human keratinocytes in response to scratch wounding in vitro. We report here that myosin II activation and recruitment to the cytoskeleton in wounded keratinocytes are biphasic. Post-wounding, a rapid phosphorylation of myosin II regulatory light chain (RLC) occurs with resultant translocation of myosin IIA to the cell cortex, far in advance of the later polarization and cell migration. During this acute-phase of myosin II activation, pharmacological approaches reveal p38-MAP kinase and cytosolic calcium as having critical roles in the phosphorylation driving cytoskeletal assembly. Although p38-MAPK has known roles in keratinocyte migration, and known roles in leading-edge focal complex dynamics, to our knowledge this is the first report of p38-MAPK acting as an upstream activator of myosin II phosphorylation and assembly during any type of wound response.     

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.     

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.     

Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-dependent process of cell membrane repair

Vesicle generation, recruitment, and exocytosis are essential for repairing disruptions of cell membranes. The functions of nonmuscle myosin IIA and IIB in this exocytotic process of membrane repair were studied by the antisense technique. Knockdown of myosin IIB suppressed wound-induced exocytosis and the membrane resealing process. Knockdown of myosin IIA did not suppress exocytosis at an initial wound and had no inhibitory effect on the resealing at initial wounds but did inhibit the facilitated rate of resealing normally found at repeated wounds made at the same site. COS-7 cells, which lack myosin IIA, did not show the facilitated response of membrane resealing to a repeated wound. S91 melanoma cells, a mutant cell line lacking myosin Va, showed normal membrane resealing and normal facilitated responses. We concluded that myosin IIB was required for exocytosis and therefore cell membrane repair itself and that myosin IIA was required in facilitation of cell membrane repair at repeated wounds. Myosin IIB was primarily at the subplasmalemma cortex and myosin IIA was concentrated at the trans-Golgi network consistent with their distinct roles in vesicle trafficking in cell membrane repair.     

Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.     

Deficiency of MTMR14 promotes autophagy and proliferation of mouse embryonic fibroblasts

MTMR14 is a phosphoinositide phosphatase, which has been reported to regulate the maintenance of normal muscle performance and aging in mice. However, the function of MTMR14 in mouse embryonic fibroblasts (MEFs) remains largely unknown. In this study, we established MTMR14 WT and KO MEFs and showed that MTMR14 is localized in whole MEFs, with higher level in nucleus and lower in cytoplasm, partially overlapping with mitochondrial. Compared with the WT control, MTMR14 KO MEFs exhibit a higher proliferation rate and more obvious autophagy. Furthermore, we demonstrate that KO of MTMR14 significantly decreased the mRNA levels of p21 and p27, while increased those of cyclinD and cyclinE. Upon (insulin-like growth factor) IGF stimulation, we also found KO of MTMR14 enhanced the phosphorylation levels of AKT and ERK in MEFs. Based on these findings, we propose that defect of MTMR14 promotes autophagy and cell proliferation in MEFs.     

Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function

Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.     

Delayed cutaneous wound closure in HO‐2 deficient mice despite normal HO‐1 expression

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO‐1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO‐2 deficient mice is impaired with exorbitant inflammation and absence of HO‐1 expression. This study addresses the role of HO‐2 in cutaneous excisional wound healing using HO‐2 knockout (KO) mice. Here, we show that HO‐2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO‐2 KO mice compared to WT controls. Surprisingly, wound closure in HO‐2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO‐1 induction in HO‐2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C‐X‐C) ligand‐11 (CXCL‐11) in wounds of HO‐2 KO mice. Abnormal regulation of CXCL‐11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL‐11 expression in HO‐2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.     

Deficiency of macrophage migration inhibitory factor gene delays healing of the medial collateral ligament: a biomechanical and biological study

The role played by macrophage migration inhibitory factor (MIF) in the process of wound healing is controversial. Besides, there have been no reports that investigated the expression or the role of MIF in the repair process after ligament injury. In this study, we hypothesized that the deficiency in MIF gene might delay ligament healing in mice. The aim of this study was to clarify this hypothesis using MIF gene-deficient mice (MIFKO) and murine model of injury to the medial collateral ligament (MCL). Biomechanical testing showed that the levels of mechanical properties were significantly lower in MIFKO than in wild-type mice (WT) on day 28 after injury. Levels of matrix metalloproteinase (MMP)-2 and -13 mRNA in the healing tissue were significantly lower in MIFKO than in WT on day 28 and on day 7, respectively. Histologically, healing tissues in MIFKO exhibited prolonged hypertrophy, poor vascularity, and prolonged increase in cell number compared with those in WT. Taken together, it was suggested that MIFKO exhibited delayed healing of the MCL, which might be caused by lower mRNA expression of MMP-2 and -13.     

GPR105 Ablation Prevents Inflammation and Improves Insulin Sensitivity in Mice with Diet-Induced Obesity

GPR105, a G protein-coupled receptor for UDP-glucose, is highly expressed in several human tissues and participates in the innate immune response. Because inflammation has been implicated as a key initial trigger for type 2 diabetes, we hypothesized that GPR105 (official gene name: P2RY14) might play a role in the initiation of inflammation and insulin resistance in obesity. To this end, we investigated glucose metabolism in GPR105 knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD). We also examined whether GPR105 regulates macrophage recruitment to liver or adipose tissues by in vivo monocyte tracking and in vitro chemotaxis experiments, followed by transplantation of bone marrow from either KO or WT donors to WT recipients. Our data show that genetic deletion of GPR105 confers protection against HFD-induced insulin resistance, with reduced macrophage infiltration and inflammation in liver, and increased insulin-stimulated Akt phosphorylation in liver, muscle, and adipose tissue. By tracking monocytes from either KO or WT donors, we found that fewer KO monocytes were recruited to the liver of WT recipients. Furthermore, we observed that uridine 5-diphosphoglucose enhanced the in vitro migration of bone marrow-derived macrophages from WT but not KO mice, and that plasma uridine 5-diphosphoglucose levels were significantly higher in obese versus lean mice. Finally, we confirmed that insulin sensitivity improved in HFD mice with a myeloid cell-specific deletion of GPR105. These studies indicate that GPR105 ablation mitigates HFD-induced insulin resistance by inhibiting macrophage recruitment and tissue inflammation. Hence GPR105 provides a novel link between innate immunity and metabolism.     

Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3 ^-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3 ^-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3 ^-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3 ^-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3 ^-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3 ^-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3 ^-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3 ^-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3 ^-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression of wound closure.     

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.