Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Neutrophil defensins enhance lung epithelial wound closure and mucin gene expression in vitro

Human airways are frequently exposed to potentially harmful agents that cause tissue injury. Upon such injury, a repair process is initiated that comprises cell migration, proliferation, and differentiation. We have previously shown that human neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) induce airway epithelial cell proliferation. Because of the role of cell proliferation in epithelial wound repair, we investigated the effect of HNP1-3 on airway epithelial wound closure and mucin gene expression in vitro. Using NCI-H292 airway epithelial cell cultures, we demonstrated that HNP1-3 cause a dose- and time-dependent increase of wound closure as well as increased cell migration. Furthermore, HNP1-3 caused a biphasic activation of the mitogen-activated protein kinase extracellular-regulated kinase 1 and 2 (ERK1/2). Both the effects of HNP1-3 on wound closure and ERK1/2 activation were blocked by specific inhibitors of the mitogen-activated protein kinase kinase MEK, whereas inhibitors of epidermal growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase, and Src did block defensin-enhanced wound closure but not ERK1/2 activation. Finally, HNP1-3 increased mRNA encoding the mucins MUC5B and MUC5AC, suggesting a role for defensins in mucous cell differentiation. These results indicate that neutrophil defensins increase epithelial wound repair in vitro, which involves migration and proliferation, and mucin production. Neutrophil defensin-enhanced wound repair appears to require epidermal growth factor receptor activation and downstream signaling pathways.     

Role of extracellular matrix and Ras in regulation of glomerular epithelial cell proliferation

Signals from extracellular matrix (ECM) to growth factor receptors regulate glomerular epithelial cell (GEC) proliferation. Epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF), or thrombin stimulated proliferation of GECs when the cells were adherent to collagen matrices, but not plastic substratum. Furthermore, EGF, HGF, or thrombin activated p42 mitogen-activated protein (MAP) kinase in collagen-adherent GECs, whereas activation was weak in GECs on plastic. To further examine the interaction of ECM with the Ras-MAP kinase cascade, GECs were stably transfected with a constitutively active Ras mutant (V12Ras). Low or moderate levels of V12Ras expression did not affect basal MAP kinase activity but, unlike parental GECs, in clones that express V12Ras, EGF was able to induce proliferation and activate MAP kinase when these cells were adherent to plastic. In parental and V12Ras-transfected GECs, MAP kinase activation was inhibited by cytochalasin D. Thus, adhesion of GECs to ECM facilitates proliferation and MAP kinase activation by mitogens acting via tyrosine kinase or non-tyrosine kinase receptors. Activation of pathway(s) downstream of V12Ras supplants signals from ECM that enable proliferation. These signals may involve the actin cytoskeleton.     

Defensins: key players or bystanders in infection, injury, and repair in the lung?

Antimicrobial peptides have been identified as key elements in the innate host defense against infection. Recent studies have indicated that the activity of antimicrobial peptides may be decreased in cystic fibrosis, suggesting a major role for these peptides in host defense against infection. One of the most intensively studied classes of antimicrobial peptides are defensins. Defensins comprise a family of cationic peptides that in human subjects can be divided into the alpha- and beta-defensin subfamilies. The alpha-defensins are produced by neutrophils and intestinal Paneth's cells, whereas beta-defensins are mainly produced by epithelial cells. Although studies on beta-defensins have so far focused on their antimicrobial activity, studies on alpha-defensins have suggested a role of these peptides in inflammation, wound repair, and specific immune responses. alpha-Defensins, which accumulate in airway secretions of patients with various chronic inflammatory lung disorders, were shown to be cytotoxic toward airway epithelial cells and to induce chemokine secretion in several cell types. Furthermore, the capacity of alpha-defensins to promote bacterial adherence to epithelial cells in vitro further supports a role for these peptides in the pathogenesis of chronic obstructive pulmonary disease and cystic fibrosis. Increased numbers of neutrophils are also present in the airways of patients with asthma, suggesting that neutrophils are involved in the pathogenesis of this disease. Because defensins are able to induce histamine release by mast cells and increase the airway hyperresponsiveness to histamine, it is tempting to speculate that defensins may also contribute to the inflammatory processes in asthma. Besides these proinflammatory effects, alpha-defensins may also display anti-inflammatory activities, including regulation of complement activation and proteinase inhibitor secretion. Finally, defensins may be involved in wound repair because defensins increase epithelial cell proliferation. Thus recent defensin research has revealed potential links between the innate and acquired immune system.     

TGF-beta suppresses EGF-induced MAPK signaling and proliferation in asthmatic epithelial cells

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.     

Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha.

Remodeling of the airways, as occurs in asthmatic patients, is associated with the continual presence of inflammatory mediators and Th2 cytokines, especially interleukin (IL)-13, during cycles of epithelial injury and repair. In this study, we examined the effect of IL-13 on well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture. IL-13 induced proliferation of NHBE cells after 24 h exposure, as reflected by [(3)H]thymidine uptake and cell counts. The effects of IL-13 were mediated through the epidermal growth factor receptor (EGFR), as proliferation was attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Proliferation appeared to be mediated by transforming growth factor (TGF)-alpha, a potent ligand for EGFR, which was released rapidly from NHBE cells in response to IL-13. Neutralizing antibody to TGF-alpha, but not antibodies against other potentially important growth factors (EGF, heparin binding epidermal growth factor-like growth factor [HB-EGF], platelet-derived growth factor [PDGF]), inhibited the mitogenic response to IL-13. This study provides the first experimental evidence that IL-13 can initiate a proliferative response of human airway epithelium in the absence of inflammatory cells or other cell types. The results are consistent with a mechanism whereby IL-13 induces release of TGF-alpha from the epithelial cells, which in turn binds via an autocrine/paracrine-type action to the EGFR, initiating proliferation. IL-13-induced airway remodeling in vivo may involve this epithelium-driven response.     

Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium

Therapy with high oxygen concentrations (hyperoxia) is often necessary to treat patients with respiratory failure. However, hyperoxia may exacerbate the development of acute lung injury, perhaps by increasing lung epithelial cell death. Therefore, interrupting lung epithelial cell death is an important protective and therapeutic strategy. In the present study, hyperoxia (95% O(2)) results in murine lung epithelium cell death by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end labeling, and Annexin V-fluorescein isothiocyanate flow cytometry assay. We show that hyperoxia increases superoxide production, as assessed by nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and flow cytometric assay, and increases phospho-extracellular signal-regulated kinase (ERK)1/2 by Western blot analysis. These processes are inhibited by a reactive oxygen species inhibitor, diphenylene iodonium (DPI), and by an inhibitor of the mitogen-activated protein (MAP) or ERK kinase (MEK)/ERK1/2 pathway, PD98059. ERK1/2 activation in hyperoxia is also inhibited by DPI. Hyperoxia-induced cell death is associated with cytochrome c release, subsequent caspase 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, which can all be suppressed by DPI and PD98059. However, the broad caspase inhibitor z-VAD-FMK protects cells from death without affecting superoxide generation and ERK1/2 activation. Taken together, our data suggest that hyperoxia, by virtue of activating NADPH oxidase, generates reactive oxygen species (ROS), which mediates cell death of lung epithelium via ERK1/2 MAPK activation, and functions upstream of caspase activation in lung epithelial cells.     

Inhibition of growth of early passage normal rat liver epithelial cell lines by epidermal growth factor

The proliferation of newly derived or early passage nonparenchymal rat liver epithelial cell lines in serum-containing medium was inhibited consistently by epidermal growth factor (EGF). Inhibition of proliferation by EGF was a dominant effect which could not be overcome by insulin, phorbol ester or substances which raised the intracytoplasmic concentration of cyclic-AMP (cholera toxin and dibutyryl cyclic-AMP). EGF has a heterogeneous effect on colony formation by individual cells of early passage cell lines. Whereas the colony-forming ability of a majority of these cells was inhibited by EGF, a small fraction of the cells resisted this effect and formed colonies in the presence of EGF. Clonal strains of EGF-inhibited and EGF-stimulated cells were isolated. Measurement of 125I-EGF binding capacity of these cell strains revealed no significant differences between the EGF-inhibited and EGF-stimulated cells. In general, EGF-stimulated cell strains demonstrated faster growth rates and higher colony-forming efficiencies than EGF-inhibited strains. EGF-induced growth inhibition however, was an unstable phenotype that might be diminished or lost after multiple passages, especially when cells were allowed to remain confluent for many days between passages. These observations may explain the consistent findings that in late passage, rat liver epithelial cell lines in which the conditions for culture and times of subcultures have not been controlled rigorously, the inhibitory effect of EGF usually is not observed.     

Increased expression of p21(waf) cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium

Because the asthmatic bronchial epithelium is characterized by widespread damage, we postulated that this is associated with expression of cell cycle inhibitors that control proliferation. Using bronchial biopsies, the epithelium was the major site of expression of the cyclin-dependent kinase inhibitor, p21(waf). Immunostaining usually occurred in the cytoplasm of columnar cells; however, in severe asthma, nuclear staining was also evident in the proliferative, basal cell compartment. p21(waf) expression was significantly higher in asthmatic versus nonasthmatic epithelium and was unaffected by corticosteroid treatment; proliferating cell nuclear antigen was not significantly different in any group. p21(waf), but not p27(kip1), mRNA and protein were induced by treatment of bronchial epithelial cells in vitro with transforming growth factor (TGF)-beta or H2O2, but not by dexamethasone, which induced p57(kip2). TGF-beta and dexamethasone inhibited epidermal growth factor (EGF)-induced DNA synthesis, whereas low concentrations of H2O2 synergized with EGF; at higher doses, growth inhibition and induction of apoptosis occurred. TGF-beta caused p21(waf) to become nuclear, suggesting interaction with the replicative machinery; however, in oxidant-stressed cells, p21(waf) was predominantly cytoplasmic, where it has been linked to cell survival. We conclude that p21(waf) overexpression in asthma influences cell proliferation and survival. This may cause abnormal repair responses that contribute to airway inflammation and remodeling.     

Interferon-gamma inhibits hepatocyte growth factor-stimulated cell proliferation of human bronchial epithelial cells: upregulation of p27(kip1) cyclin-dependent kinase inhibitor.

Proliferation of bronchial epithelial cells is an important biologic process in a variety of physiologic and pathologic conditions. In this study, we demonstrate that hepatocyte growth factor (HGF) stimulates proliferation of human bronchial epithelial cells obtained from healthy volunteers. The mitogenic effect of HGF is dependent on costimulation with serum and is completely abrogated by interferon-gamma (IFN-gamma). In the absence of serum, HGF is capable of inducing activation of extracellular signal-regulated kinases (ERK)1 and ERK2, but fails to stimulate proliferation by itself. These effects of HGF and IFN-gamma were reproduced faithfully in BEAS-2B cells, which are an immortalized cell line derived from human bronchial epithelial cells. Further, we investigated the molecular mechanisms underlying the effects of HGF and IFN-gamma in BEAS-2B cells and found that the MEK1 inhibitor PD98059, but not the p38 M-associated protein kinase inhibitor SB203580, abrogates HGF-induced ERK activation and proliferation in response to HGF and serum. In addition, LY294002, which is the specific inhibitor of phosphatidyl inositol 3-kinase, partially inhibited HGF- and serum-stimulated proliferation. We also found that HGF by itself is capable of inducing a G1 cyclin, cyclin D1, but fails to downregulate p27(kip1) cyclin-dependent kinase (CDK) inhibitor, which is a requisite for G1 to S phase cell cycle progression. IFN-gamma does not interfere with the effects of HGF on either ERK activation or cyclin D1 induction; however, it prevents the downregulation of p27(kip1) CDK inhibitor that takes place in response to a combination of HGF and serum. These results indicate that the MEK-ERK signaling pathway is necessary but not sufficient for human bronchial epithelial cell proliferation, and implicate the significance of HGF and IFN-gamma in the repair processes of injured human bronchial epithelial cells.     

Involvement of mitogen-activated protein kinase in transforming growth factor alpha-stimulated cell proliferation in the cultured granulosa cells of the Japanese quail

The avian granulosa cells proliferate during follicular growth phase and differentiate to produce progesterone in response to luteinizing hormone (LH) when the follicle becomes the largest. In order to study the involvement of mitogen-activated protein (MAP) kinase in proliferation of the granulosa cells in avian species, quail granulosa cells were cultured for 66 h with various hormones (follicle stimulating hormone (FSH), LH, progesterone, estradiol-17beta, testosterone), or growth factors (transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), IGF-II), and the presence of immunodetectable MAP kinase was examined in the cell lysates. When the granulosa cells were cultured with TGF alpha, the cell number as well as the incorporation of [3H]thymidine was increased. Other hormones or growth factors caused no significant increase in cell numbers. Stimulation of the cells with TGF alpha for 10 min caused a retarded mobility of MAP kinase in the gel of SDS-PAGE. Both the increases in [3H]thymidine incorporation and the retarded mobility were inhibited by the presence of a tyrosine kinase inhibitor, genistein, indicating the importance of phosphorylation of protein during the TGF alpha-stimulation.     

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.     

Extracellular signal-regulated kinase activation delays hyperoxia-induced epithelial cell death in conditions of Akt downregulation

Hyperoxia (fraction of inspired oxygen = 95%) induces death of lung epithelial cells. The duration of cell survival in the setting of hyperoxia depends on hyperoxia-induced activation of intracellular survival pathways. Two survival pathways with known effects on lung epithelial cells are the propidium iodide 3-kinase/Akt and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathways. We investigated the effect of hyperoxia on activity of both the Akt and ERK pathways in the A549 lung epithelial cell line. Hyperoxia-exposed cells show progressive loss of Akt activation and total Akt protein. Hyperoxia decreases Akt mRNA, consistent with the loss of total Akt. In addition, hyperoxia induces ERK activation. Inhibition of ERK with the MAP kinase kinase 1/2 inhibitor, U0126, shortens the survival time of cells in hyperoxia, suggesting that increased ERK activity partially compensates for the hyperoxia-induced Akt downregulation. Our findings show, for the first time, that hyperoxia has divergent effects on two survival pathways (Akt and ERK), and that ERK activity compensates for the loss of the Akt survival effects, delaying the death of hyperoxia-exposed lung epithelial cells.     

Src Tyrosine Kinase Inhibitor,M475271, Suppresses Subcutaneous Growth and Production of Lung Metastasis Via Inhibition of Proliferation,Invasion, and Vascularization of Human Lung Adenocarcinoma Cells

Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of all three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.     

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.     

In vivo and in vitro assessment of mitogen activated protein kinase involvement during quail secondary palate formation

Spatiotemporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate. An understanding of the mechanisms by which these cellular phenomena are regulated during palate development involves the identification of the various signal transduction pathways. In the present study, the presence and activation of mitogen-activated protein (MAP) kinases were investigated during the development of quail secondary palate. The palatal shelves were dissected on days 5–9 of incubation, homogenized, and centrifuged, after which the samples were separated by anion exchange fast protein liquid chromatography. The fractions were analyzed for myelin basic protein (MBP) phosphorylation. In addition, primary cultures of quail palate mesenchymal cells (QPMCs) were treated with epidermal growth factor (EGF) and prepared for MBP phosphorylation assays. A temporally regulated pattern of phosphotransferase activity, characterized by a three-fold increase in phosphotransferase activity toward MBP between days 5 and 8 of incubation, was observed during quail palate development. Western blotting, using MAP kinase antibodies, demonstrated the presence of a 42-kDa isoform between days 5 and 9 of incubation, during which the level of protein remained constant. Antityrosine immunoblotting with 4G10 also detected a 42-kDa protein. Phosphotransferase assays, using either a MAP kinase-specific substrate peptide (S5) or a protein kinase C inhibitor (R3), further confirmed the presence of a MAP kinase in the developing palate of quail. Because diverse biological processes occur concurrently during in vivo palate morphogenesis, the involvement of MAP kinase was explored further in primary cell culture. The data showed that EGF stimulated proliferation and activated 42-kDa MAP kinase in QPMCs. It is suggested that MAP kinase cascade may be involved in growth factor-regulated cell proliferation during morphogenesis of quail secondary palate. Anat. Rec. 252:194–204, 1998. © 1998 Wiley-Liss, Inc.     

p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts

Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor-induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.