[Show abstract][Hide abstract] ABSTRACT: Following injury, keratinocytes switch gene expression programs from the one that promotes differentiation to the one that supports migration. A common feature of human wounds and ulcerations of any form is the expression of matrix metalloproteinase 1 (MMP-1; collagenase-1) by leading-edge basal keratinocytes migrating across the dermal or provisional matrix. Induction of MMP-1 occurs by signaling from the α2β1 integrin in contact with dermal fibrillar type I collagen, and the activity of MMP-1 is required for human keratinocytes to migrate on collagen. Thus, MMP-1 serves a critical role in the repair of damaged human skin. Here, we evaluated the mechanisms controlling MMP-1 expression in primary human keratinocytes from neonatal foreskin and adult female skin. Our results demonstrate that shortly following contact with type I collagen extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase were markedly activated, whereas c-Jun N-terminal kinase (JNK) phosphorylation remained at basal levels. ERK inhibition markedly blocked collagen-stimulated MMP-1 expression in keratinocytes. In contrast, inhibiting p38 or JNK pathways had no effect on MMP-1 production. Moreover, investigating the role of Rho GTPases revealed that Cdc42 attenuates MMP-1 expression by suppressing ERK activity. Thus, our data indicate that injured keratinocytes induce MMP-1 expression through ERK activation, and this process is negatively regulated by Cdc42 activity.Journal of Investigative Dermatology advance online publication, 19 December 2013; doi:10.1038/jid.2013.499.
Journal of Investigative Dermatology 11/2013; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis.
We first examined mice that express alphaSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFbeta on expression of markers of myofibroblast activation in vitro, including alphaSMA, collagen alpha1, type 1, collagen alpha1, type III, and TGFbeta expression.
After bleomycin injury, we found decreased number of alphaSMA-GFP + cells in A12 treated mice, validated by alphaSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-beta, did not affect alphaSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of alphaSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on alphaSMA stress fiber organization in mouse lung fibroblasts.
Respiratory research 10/2013; 14(1):102. · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Airway epithelial cells cultured at an air-liquid interface bear many hallmarks of in vivo cells and are used extensively to study the biology of the lung epithelium. Because miRNAs regulate many cellular functions, we postulated that miRNA profiling would provide an unbiased assessment of the effects of in vitro culturing. RNA was extracted from primary airway epithelial cells either immediately after cell procurement (in vivo condition) or after air-liquid interface culture was established (in vitro condition). We assessed 742 miRNAs and determined differential expression between in vivo and in vitro conditions. Air-liquid interface culturing of airway epithelial cells caused widespread changes in miRNA expression. A similarly extensive alteration in gene expression was observed in an independent set of publicly available microarray data. We integrated miRNA and gene expression results to identify culture-induced differences in transcriptional programs (including several involved in epithelial injury and repair). Air-liquid interface cultures are useful models for studying airway biology, but the present findings indicate that, despite phenotypic similarities with primary cells, these culture systems profoundly perturb miRNA and gene expression. Studies of lung epithelium based on in vitro culture should therefore be designed and interpreted with an appreciation of the limitations of air-liquid interface culture systems.
American Journal Of Pathology 08/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase 7 (MMP7) expression is quickly upregulated after injury and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild type and Mmp7-null mice cultured at an air-liquid interface. Analysis of differentially expressed genes between genotypes after injury revealed enrichment of functional categories associated with inflammation, cilia and differentiation. Because these analyses suggested MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production in Mmp7-null compared to wild type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.
American Journal of Respiratory Cell and Molecular Biology 12/2012; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetracyclines are broad-spectrum antibiotics that are also used to induce gene expression using the reverse tetracycline transactivator / tetracycline operator system (rtTA/tetO system). The system assumes that tetracyclines have no effects on mammals. However, a number of studies suggest that tetracyclines may have powerful anti-inflammatory effects. We report that the tetracycline, doxycycline, inhibits neutrophil (PMN) influx into the lungs of mice treated with bacterial endotoxin (LPS).
Mice were challenged with intratracheal LPS in the presence or absence of doxycyline. bronchoalveolar lavage cell counts and differential, total bronchoalveolar lavage protein, lung homogenate caspase-3 and tissue imaging were used to assess lung injury. In addition, PMN chemotaxis was measured in vitro and syndecan-1 was measured in bronchoalveolar lavage fluid.
The administration of doxycycline resulted in a significant decrease in the number of bronchoalveolar lavage PMNs in LPS-treated mice. Doxycycline had no effect on other markers of lung injury such as total bronchoalveolar lavage protein and whole lung caspase-3 activity. However, doxycycline resulted in a decrease in shed syndecan-1 in bronchoalveolar lavage fluid.
We conclude that doxycycline has an important anti-inflammatory effect that can potentially confound the experiments in which the rtTA/tetO system is being used to study the immune response.
Journal of Inflammation 09/2012; 9(1):31. · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Syndecan-1 is a cell surface proteoglycan that can organize co-receptors into a multimeric complex to transduce intracellular signals. The syndecan-1 core protein has multiple domains that confer distinct cell- and tissue-specific functions. Indeed, the extracellular, transmembrane, and cytoplasmic domains have all been found to regulate specific cellular processes. Our previous work demonstrated that syndecan-1 controls lung epithelial migration and adhesion. Here, we identified the necessary domains of the syndecan-1 core protein that modulate its function in lung epithelial repair. We found that the syndecan-1 transmembrane domain has a regulatory function in controlling focal adhesion disassembly, which in turn controls cell migration speed. In contrast, the extracellular domain facilitates cell adhesion through affinity modulation of α(2)β(1) integrin. These findings highlight the fact that syndecan-1 is a multidimensional cell surface receptor that has several regulatory domains to control various biological processes. In particular, the lung epithelium requires the syndecan-1 transmembrane domain to govern cell migration and is independent from its ability to control cell adhesion via the extracellular domain.
Journal of Biological Chemistry 08/2012; 287(42):34927-35. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After injury, residual epithelial cells coordinate contextual clues from cell-cell and cell-matrix interactions to polarize and migrate over the wound bed. Protrusion formation, cell body translocation and rear retraction is a repetitive process that allows the cell to move across the substratum. Fundamental to this process is the assembly and disassembly of focal adhesions that facilitate cell adhesion and protrusion formation. Here, we identified syndecan-1 as a regulator of focal adhesion disassembly in migrating lung epithelial cells. Syndecan-1 altered the dynamic exchange of adhesion complex proteins, which in turn regulates migration speed. Moreover, we provide evidence that syndecan-1 controls this entire process through Rap1. Thus, syndecan-1 restrains migration in lung epithelium by activating Rap1 to slow focal adhesion disassembly.
Journal of Cell Science 08/2012; · 5.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vitamin D deficiency is increasing in incidence around the world. Vitamin D, a fat-soluble vitamin, has documented effects on the innate and adaptive immune system, including macrophage and T regulatory (Treg) cell function. Since Treg cells are important in acute lung injury resolution, we hypothesized that vitamin D deficiency increases the severity of injury and delays injury resolution in lipopolysaccharide (LPS) induced acute lung injury. Vitamin D deficient mice were generated, using C57BL/6 mice, through diet modification and limited exposure to ultraviolet light. At 8 weeks of age, vitamin D deficient and sufficient mice received 2.5 g/kg of LPS or saline intratracheal. At 1 day, 3 days and 10 days, mice were anesthetized and lung elastance measured. Mice were euthanized and bronchoalveolar lavage fluid, lungs and serum were collected. Ex vivo neutrophil chemotaxis was evaluated, using neutrophils from vitamin D sufficient and deficient mice exposed to the chemoattractants, KC/CXCL1 and C5a, and to bronchoalveolar lavage fluid from LPS-exposed mice. We found no difference in the degree of lung injury. Leukocytes were mildly decreased in the bronchoalveolar fluid of vitamin D deficient mice at 1 day. Ex-vivo, neutrophils from vitamin D deficient mice showed impaired chemotaxis to KC but not to C5a. Vitamin D deficiency modestly impairs neutrophil chemotaxis; however, it does not affect lung injury or its resolution in an LPS model of acute lung injury.
PLoS ONE 01/2012; 7(11):e49076. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Supplemental O(2) is commonly employed in patients with respiratory failure; however, hyperoxia is also a potential contributor to lung injury. In animal models, hyperoxia causes oxidative stress in the lungs, resulting in increased inflammation, edema, and permeability. We hypothesized that oxidative stress from prolonged hyperoxia leads to endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) and induction of CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor associated with cell death in the setting of persistent ER stress. To test this hypothesis, we exposed the mouse lung epithelial cell line MLE-12 to 95% O(2) for 8-24 h and evaluated for evidence of UPR induction and CHOP induction. Hyperoxia caused increased CHOP expression without other evidence of UPR activation. Because CHOP expression is preceded by phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α), we evaluated the role of double-stranded RNA-activated protein kinase (PKR), a non-UPR-associated eIF2α kinase. Hyperoxia caused PKR phosphorylation, and RNA interference knockdown of PKR attenuated hyperoxia-induced CHOP expression. In vivo, hyperoxia induced PKR phosphorylation and CHOP expression in the lungs without other biochemical evidence for ER stress. Additionally, Ddit3(-/-) (CHOP-null) mice had increased lung edema and permeability, indicating a previously unknown protective role for CHOP after prolonged hyperoxia. We conclude that hyperoxia increases CHOP expression via an ER stress-independent, PKR-dependent pathway and that increased CHOP expression protects against hyperoxia-induced lung injury.
[Show abstract][Hide abstract] ABSTRACT: In response to injury, epithelial cells migrate across the denuded tissue to rapidly close the wound and restore barrier, thereby preventing the entry of pathogens and leakage of fluids. Efficient, proper migration requires a range of processes, acting both inside and out of the cell. Among the extracellular responses is the expression of various matrix metalloproteinases (MMPs). Though long thought to ease cell migration simply by breaking down matrix barriers, findings from various models demonstrate that MMPs facilitate (and sometimes repress) cell movement by other means, such as affecting the state of cell-matrix interactions or proliferation. In this Prospect, we review some key data indicting how specific MMPs function via their activity as proteinases to control closure of epithelial wounds.
Journal of Cellular Biochemistry 10/2009; 108(6):1233-43. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lung injury promotes the expression of matrix metalloproteinase-7 (MMP7, matrilysin), which is required for neutrophil recruitment and re-epithelialization. MMP7 governs the lung inflammatory response through the shedding of syndecan-1. Because inflammation and repair are related events, we evaluated the role of syndecan-1 shedding in lung re-epithelialization.
Epithelial injury induced syndecan-1 shedding from wild-type epithelium but not from Mmp7(-/-) mice in vitro and in vivo. Moreover, cell migration and wound closure was enhanced by MMP7 shedding of syndecan-1. Additionally, we found that syndecan-1 augmented cell adhesion to collagen by controlling the affinity state of the alpha(2)beta(1) integrin.
MMP7 shedding of syndecan-1 facilitates wound closure by causing the alpha(2)beta(1) integrin to assume a less active conformation thereby removing restrictions to migration. MMP7 acts in the lungs to regulate inflammation and repair, and our data now show that both these functions are controlled through the shedding of syndecan-1.
[Show abstract][Hide abstract] ABSTRACT: Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.
American Journal Of Pathology 06/2008; 172(5):1256-70. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Obliterative bronchiolitis (OB) is a major cause of allograft dysfunction after lung transplantation and is thought to result from immunologically mediated airway epithelial destruction and luminal fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the regulation of lung inflammation, airway epithelial repair, and extracellular matrix remodeling and therefore may participate in the pathogenesis of OB. The goals of this study were to determine the expression profiles of MMPs and TIMPs and the role of TIMP-1 in the development of airway obliteration using the murine heterotopic tracheal transplant model of OB. We demonstrate the selective induction of MMP-3, MMP-9, MMP-12, and TIMP-1 in a temporally restricted manner in tracheal allografts compared with isografts. In contrast, the expression of MMP-7, TIMP-2, and TIMP-3 was decreased in allografts relative to isografts during the period of graft rejection. TIMP-1 protein localized to epithelial, mesenchymal, and inflammatory cells in the tracheal grafts in a temporally and spatially restricted manner. Using TIMP-1-deficient mice, we demonstrate that the absence of TIMP-1 in the donor trachea or the allograft recipient reduced luminal obliteration and increased re-epithelialization in the allograft compared with wild-type control at 28 d after transplantation. Our findings provide direct evidence that TIMP-1 contributes to the development of airway fibrosis in the heterotopic tracheal transplant model, and suggest a potential role for this proteinase inhibitor in the pathogenesis of OB in patients with lung transplant.
American Journal of Respiratory Cell and Molecular Biology 05/2006; 34(4):464-72. · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bleomycin-induced lung injury triggers a profound and durable increase in tissue inhibitor of metalloproteinase (TIMP)-1 expression, suggesting a potential role for this antiproteinase in the regulation of lung inflammation and fibrosis. TIMP-1 protein induction is spatially restricted to areas of lung injury as determined by immunohistochemistry. Using TIMP-1 null mutation mice, we demonstrate that TIMP-1 deficiency amplifies acute lung injury as determined by exaggerated pulmonary neutrophilia, hemorrhage, and vascular permeability compared with wild-type littermates after bleomycin exposure. The augmented pulmonary neutrophilia observed in TIMP-1-deficient animals was not found in similarly treated TIMP-2-deficient mice. Using TIMP-1 bone marrow (BM) chimeric mice, we observed that the TIMP-1-deficient phenotype was abolished in wild-type recipients of TIMP-1-deficient BM but not in TIMP-1-deficient recipients of wild-type BM. Acute lung injury in TIMP-1-deficient mice was accompanied by exaggerated gelatinase-B activity in the alveolar compartment. TIMP-1 deficiency did not alter neutrophil chemotactic factor accumulation in the injured lung nor neutrophil migration in response to chemotactic stimuli in vivo or in vitro. Moreover, TIMP-1 deficiency did not modify collagen accumulation after bleomycin injury. Our results provide direct evidence that TIMP-1 contributes significantly to the regulation of acute lung injury, functioning to limit inflammation and lung permeability.
American Journal of Respiratory Cell and Molecular Biology 10/2005; 33(3):271-9. · 4.11 Impact Factor