Lysophosphatidic acid stimulates epidermal growth factor‐family ectodomain shedding and paracrine signaling from human lung fibroblasts

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase‐tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme‐linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase‐tagged heparin‐binding epidermal growth factor (HB‐EGF), amphiregulin, and transforming growth factor‐a; non‐transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB‐EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal‐regulated kinase 1/2, enhanced expression of mRNA for c‐fos, HB‐EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA‐stimulated cells, and found enhanced EGFR and extracellular signal‐regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF‐family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.     

093Prostaglandin E2 Inhibits Fibroblast Migration: Implications for Wound Healing

Background : Wound healing is a complex process involving multiple cell types, extracellular matrix components and soluble mediators. Prostaglandin E2 is an important component of the inflammatory response to injury. PGE2 can regulate the fibroblast response to injury via the EP receptor family. Here, we examine PGE2 regulation of fibroblast migration. Our analysis extends to fibroblasts representing a spectrum of wound healing phenotypes. Hypothesis : Prostaglandin E2 mediated inhibition of fibroblast migration is conserved across multiple fibroblast phenotypes. Methods : Primary cultures of human fetal, adult and keloid fibroblasts were used. Analysis of the EP receptor profile for each fibroblast phenotype was conducted using real‐time PCR, Western blot and immunohistochemistry. Fibroblast migration was quantified using a well established in vitro scratch assay. Results : Prostaglandin E2, via EP2/EP4 receptors, inhibits fibroblast migration in all fibroblast phenotypes. Fetal fibroblasts retain a more robust migratory phenotype when compared to normal adult and keloid fibroblasts. Normal adult fibroblasts exhibit a dramatic destabilization of the actin cytoskeleton which accompanies PGE2 inhibition of cell migration. This effect was not observed in fetal or keloid fibroblasts. Conclusions : Fibroblast activity in the wound bed can be altered by inflammatory mediators. The effects of prostaglandin E2 appear to be partially conserved across various fibroblast phenotypes. Variability in the response of these cells, however, indicates that fibroblasts derived from fetal tissue may retain intrinsic altered response mechanisms to endogenous inflammatory mediators.     

Epidermal growth factor protects fibroblasts from apoptosis via PI3 kinase and Rac signaling pathways

The fibroplasia noted during wound repair is resolved by fibroblast cell death. How fibroblasts undergo death and how this is prevented by trophic growth factors present during the regenerative phase are unknown at the molecular level. We examined a model of staurosporine-induced apoptosis in fibroblasts. We demonstrated that epidermal growth factor (EGF) stimulation of fibroblast NR6WT expressing human EGF receptors blocks staurosporine-induced apoptosis by inhibiting the activation of caspase-3. The survival effect of EGF on rescuing apoptotic NR6WT involves signaling pathways that derive from PI3K and Rac; the blockade of apoptosis is abolished when PI3K and Rac signals are inhibited simultaneously. Furthermore, by using KP372-1, a specific Akt inhibitor, we found that downstream of Akt signaling pathways is absolutely required for the EGF rescue from staurosporine-induced apoptosis in NR6WT. Interestingly, EGF prevention of apoptosis induced by tumor necrosis factor-α in the face of cycloheximide blockade of protein translation occurs via a different set of pathways as the simultaneous inhibition of extracellular signal-regulated kinase, Rac, and PI3K signaling did not eliminate EGF from rescuing fibroblasts in the face of this cytokine. These findings indicate that EGF receptor activation provides survival response against staurosporine-induced apoptosis through signal pathways of PI3K and Rac, which then may prevent the activation of caspase-3.     

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway

BACKGROUND: Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. CONCLUSIONS: Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.     

Desmoid cell motility is induced in vitro by rhEGF

Desmoid tumors are benign but locally invasive myofibroblastic lesions that arise predominantly in the abdominal wall or shoulder and are prone to aggressive local recurrences. A perceived association between desmoid activity and the expression of growth factors during pregnancy or following trauma suggests a cause‐and‐effect relationship between growth factor stimulation and desmoid invasiveness. We used Boyden Chambers to quantify cell motility in order to determine the effect of growth factor stimulation on desmoid cell migration. Desmoid cell cultures were treated under serum‐free conditions with epidermal growth factor (rhEGF) or transforming growth factor alpha (rhTGFα). Additional cell cultures were pretreated under serum‐free conditions with the EGF receptor (EGFR) inhibitor AG1478, alone or in combination with the TGFβ1 receptor inhibitor SB431542, and then stimulated with growth factor prior to being assayed for cell motility. The experiments demonstrated a direct dose‐dependent relationship between rhEGF stimulation and desmoid motility. In contrast, rhTGFα was less effective at inducing cell migration. rhEGF‐induced cell migration could be diminished, but not reduced to control levels, by inhibiting EGFR. When EGF and TGFβ1 receptors were inhibited simultaneously, the level of rhEGF‐induced cell migration was reduced significantly beyond the level of cell migration generated by inhibition of EGFR alone. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor

BACKGROUND: Angiotensin II (Ang II) is a potent stimulus of vascular smooth muscle cell (VSMC) growth. Activation of extracellular signal-regulated kinase (ERK), the archetypal mitogen-activated protein (MAP) kinase, and phosphatidylinositol 3 (PI3) kinase are critical steps in Ang II-induced mitogenic signaling. However, the mechanism involved in the activation of these kinases upon binding of Ang II to its receptor is poorly understood. METHODS: In the present study, we examined the role of the epidermal growth factor receptor (EGFR) in Ang II signaling in VSMCs employing immunoprecipitation, Western blot analysis, kinase immunocomplex assay, and [3H]-thymidine incorporation. RESULTS: A time-dependent tyrosine phosphorylation of the EGFR in response to Ang II was observed that was mediated by the Ang II type 1 receptor. This transactivation of the EGFR was blocked in the presence of PP1, an inhibitor of the intracellular Src-like tyrosine kinases. The tyrphostin AG 1478, a selective EGFR antagonist, inhibited both Ang II- and EGF-induced tyrosine phosphorylation of the EGFR. Furthermore, Ang II induced the binding of the adaptor protein Shc to the EGFR, leading to phosphorylation of Shc. In addition, the same nanomolar concentrations of AG 1478 that were effective in EGF signaling blocked the Ang II-induced activation of ERK and PI3 kinase in a dose-dependent manner. Proliferation of VSMCs, detected by measurements of DNA synthesis, following stimulation with Ang II was potently inhibited in the presence of AG 1478 or PP1. CONCLUSION: Our data suggest that EGFR serves as a role in mitogenic signaling following stimulation with Ang I through a ligand-independent and Src-dependent transactivation of the EGFR. Furthermore, we demonstrate this transactivation as a pivotal step in Ang II-induced activation of MAP kinase and PI3 kinase, as well as growth of VSMCs.     

Activation of ERK and p38 kinase mediated keloid fibroblast apoptosis after flashlamp pulsed-dye laser treatment

BACKGROUND AND OBJECTIVES: Flashlamp pulsed-dye lasers (PDLs) revealed effective regression or arrest in patients with keloids in our clinical studies [Kuo YR et al., Laser Surg Med 2004;34:104-108]. In this study, we further investigated whether the induction of keloid regression seen with PDL treatment through activation in mitogen-activated protein (MAP) kinase and caspase promotes cell apoptosis and reduces fibroblast proliferation. STUDY DESIGN/MATERIALS AND METHODS: Keloid tissues were obtained from 10 patients with intralesional or punch biopsies prior to and 7 days after PDL treatments [fluence per pulse was 10-18 J/cm2 (mean 14 J/cm2)]. Prior to and after PDL treatments, the proliferating fibroblasts in keloid tissue were immunohistochemically detected by proliferating cell nuclear antigen (PCNA) expression. The apoptotic cell was detected by terminal deoxynucleotidyl transferase dUTP-nick end labeling (TUNEL) staining and fragmented caspase-3 expression. MAP kinase activation as represented by extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK) expression of keloid tissues was investigated by immunohistochemical (IHC) staining, respectively. RESULTS: IHC staining indicated that PCNA expression of fibroblasts was significantly reduced in keloid tissue after PDL irradiation. TUNEL assay revealed lower apoptotic cells expression in the keloid tissue prior to laser treatment. Following laser treatment, apoptotic cells with relatively strong DNA damage and fragmentation were seen in all keloid biopsy samples, especially in the keloid fibroblast population. The activation of ERK and p38 MAP kinase increased significantly in keloid tissue after PDL treatment. JNK was shown to be unchanged. CONCLUSIONS: The PDL treatment is shown to induce keloid regression through suppression of keloid fibroblast proliferation, induction of apoptosis, and upregulation of ERK and p38 MAP kinase activity.     

Molecular dissection of abnormal wound healing processes resulting in keloid disease

Keloids are locally aggressive scars that typically invade into healthy surrounding skin and cause both physical and psychosocial distress to the patient. These pathological scars occur following minimal skin trauma after a variety of causes including burns and trauma. Although the pathogenesis of keloid disease is not well understood, it is considered to be the end product of an abnormal healing process. The aim of this review was to investigate the molecular and cellular pathobiology of keloid disease in relation to the normal wound healing process. The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups: (1) extracellular matrix proteins and their degradation, (2) cytokines and growth factors, and (3) apoptotic pathways. With respect to cellular involvements, fibroblasts are the most well‐studied cell population. However, it is unclear whether the fibroblast is the causative cell; they are modulated by other cell populations in wound repair, such as keratinocytes and macrophages. This review presents a detailed account of individual phases of the healing process and how they may potentially be implicated in aberrant raised scar formation, which may help in clarifying the mechanisms involved in keloid disease pathogenesis.     

瘢痕疙瘩成纤维细胞的基因组学研究

目的 寻找瘢痕疙瘩致病相关基因，探讨瘢痕疙瘩的发生机理。方法 利用含1100个人类肿瘤相关基因的cDNA芯片(cDNA—microarray)对耳垂和胸部瘢痕疙瘩及正常皮肤成纤维细胞进行检测，初步分析瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞基因总体表达的差异，并筛选出差异基因。结果 在耳垂及胸部瘢痕疙瘩成纤维细胞中，分别有8种和17种特异性表达基因被检出。在正常皮肤中特异性表达的细胞增殖抑制基因Mda-7，在耳垂及胸部瘢痕疙瘩成纤维细胞中均未被表达。结论 多种基因参与了瘢痕疙瘩的形成过程，瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞之间存在基因表达的差异，增殖因子受体PAR-1和增殖抑制基因Mda-7可能参与瘢痕疙瘩的形成。     

Perturbation of cell cycle expression in keloid fibroblast

The pathogenesis of keloid formation is poorly understood. The fibroblasts in keloid patients continue to multiply even after initial wound repair and are characterized by a persistent dermal fibroproliferative reaction and excessive extracellular matrix production. Most studies concentrate on the type of collagen produced within keloids and the cytokines that dominate the disease. There have been considerably fewer studies in the expression of messenger RNA level in key cell cycle genes of the keloid fibroblast. The aim of this study was to measure the messenger RNA expression of the key regulators of cell cycle, cell cycle cyclins, and cyclin-dependent kinases, and their inhibitors.     

Role and mechanism of Circ-PDE7B in the formation of keloid

The excessive proliferation of keloid fibroblasts is one of the important reasons leading to the formation of keloids. Circular RNA (circRNA) is an important regulator that regulates the biological functions of cells. However, the role and mechanism of circ-PDE7B in keloid formation have not been studied yet. QRT-PCR was used to detect the circ-PDE7B, miR-331-3p and cyclin-dependent kinase 6 (CDK6) expression. The biological functions of keloid fibroblasts were determined by MTT assay, flow cytometry, transwell assay and wound healing assay. Western blot analysis was used to measure the protein levels of extracellular matrix (ECM) markers and CDK6. The interaction between miR-331-3p and circ-PDE7B or CDK6 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-PDE7B was found to be upregulated in keloid tissues and fibroblasts. Downregulation of circ-PDE7B could suppress the proliferation, invasion, migration, ECM accumulation and accelerate the apoptosis of keloid fibroblasts. Circ-PDE7B could serve as a sponge of miR-331-3p, and the regulation of silenced circ-PDE7B on the biological functions of keloid fibroblasts could be abolished by miR-331-3p inhibitor. Additionally, CDK6 was a target of miR-331-3p, and its overexpression could reverse the negative regulation of miR-331-3p on the biological functions of keloid fibroblasts. Circ-PDE7B sponged miR-331-3p to positively regulate CDK6 expression. Taken together, circ-PDE7B promoted the proliferation, invasion, migration and ECM accumulation of keloid fibroblasts by regulating the miR-331-3p/CDK6 axis, suggesting that circ-PDE7B might be a potential target for keloid treatment.     

Calcium-sensing receptor activation stimulates parathyroid hormone-related protein secretion in prostate cancer cells: role of epidermal growth factor receptor transactivation

We have previously reported that high extracellular Ca2+ stimulates parathyroid hormone-related protein (PTHrP) release from human prostate and breast cancer cell lines as well as from H-500 rat Leydig cancer cells, an action mediated by the calcium-sensing receptor (CaR). Activating the CaR leads to phosphorylation of mitogen-activated protein kinases (MAPKs) that participate in PTHrP synthesis and secretion. Because the CaR is a G protein-coupled receptor (GPCR), it is likely to transactivate the epidermal growth factor receptor (EGFR) or the platelet-derived growth factor receptor (PDGFR). In this study, we hypothesized that activation of the CaR transactivates the EGFR or PDGFR, and examined whether transactivation affects PTHrP secretion in PC-3 human prostate cancer cells. Using Western analysis, we observed that an increase in extracellular Ca2+ resulted in delayed activation of extracellular signal-regulated kinase (ERK) in PC-3 cells. Pre-incubation with AG1478 (an EGFR kinase inhibitor) or an EGFR neutralizing antibody inhibited the high Ca2+ -induced phosphorylation of ERK1/2. GM6001, a pan matrix metalloproteinase (MMP) inhibitor, also partially suppressed the ERK activation, but AG1296 (a PDGFR kinase inhibitor) did not. High extracellular Ca2+ stimulates PTHrP release during a 6-h incubation (1.5- to 2.5- and 3- to 4-fold increases in 3.0 and 7.5 mM Ca2+, respectively). When cells were preincubated with AG1478, GM6001, or an antihuman heparin-binding EGF (HB-EGF) antibody, PTHrP secretion was significantly inhibited under basal as well as high Ca2+ conditions, while AG1296 had no effect on PTHrP secretion. Taken together, these findings indicate that activation of the CaR transactivates the EGFR, but not the PDGFR, leading to phosphorylation of ERK1/2 and resultant PTHrP secretion, although CaR-EGFR-ERK might not be the only signaling pathway for PTHrP secretion. This transactivation is most likely mediated by activation of MMP and cleavage of proheparin-binding EGF (proHB-EGF) to HB-EGF.     

Lidocaine inhibits tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells

BACKGROUND: Although lidocaine is recognized as an excellent topical corneal analgesic, its toxic effect on corneal epithelial cells limits its use during corneal epithelial wound healing. Mechanism of the impairment of corneal reepithelialization with lidocaine, however, has not been evaluated. The authors' previous study revealed that lidocaine inhibits the activity of tyrosine kinase receptors through the interaction with specific amino acid sequences around autophosphorylation sites, including acidic, basic, and aromatic amino acids. Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with an important role in epithelial cell proliferation after corneal wounding, also possesses these amino acids sequences around autophosphorylation sites. The authors hypothesized that lidocaine would suppress tyrosine kinase activity of EGFR and would impair corneal epithelial cell proliferation. METHODS: To investigate the effect of lidocaine (4 microM-40 mM) on epidermal growth factor (EGF)-stimulated autophosphorylation of EGFR, the authors studied purified EGFR in microtubes. They cultured human corneal epithelial cells (HCECs) with EGF and lidocaine to investigate the effect of lidocaine on cell proliferation and on autophosphorylation of EGFR in HCECs. RESULTS: Lidocaine (> or =400 microM) significantly suppressed EGF-stimulated autophosphorylation of the purified EGFR. In the HCEC study, EGF alone stimulated cell proliferation and increased autophosphorylation of EGFR in HCECs. Lidocaine (> or = 400 microM) significantly suppressed both the proliferation of HCECs promoted by EGF and EGF-stimulated autophosphorylation of EGFR. CONCLUSION: Lidocaine directly inhibits tyrosine kinase activity of EGFR and suppresses the corneal epithelial cell proliferation.     

Role of protein kinase a pathway in epidermal growth factor-induced liver cell repair

To gain a better understanding of the mechanisms that control the repair process in the injured liver, the actions of epidermal growth factor (EGF) and protein kinase A (PKA) were studied. Normal rat liver cells (clone 9) were grown to confluence. Standardized excisional wounds were made with a razor blade. The extent of hepatocyte migration into the wound was measured and determined at specific time intervals using a computerized digital analyzing system. Immunostaining of F-actin was performed with a fluorescein-labeled phalloidin. EGF significantly stimulated liver cell migration, whereas specific EGF-neutralizing antibody inhibited the EGF-induced migration. Agents that activate PKA at different stages of the PKA activation pathway, including 3-isobutyl-1-methylxanthine (IBMX), forskolin, and cholera toxin, inhibited EGF-induced migration. EGF triggered formation of actin stress fibers. PKA-activating agents inhibited actin stress fiber formation and stretching of cells at the wound margin. The following conclusions were drawn: (1) In excisional wounds of hepatocyte monolayers, both EGF and PKA exert action on actin microfilaments, which are stretched by EGF and inhibited by PKA; (2) the enhanced repair of wounded hepatocyte monolayers by EGF is blocked by activation of the PKA pathway at various levels; and (3) these actions of EGF and PKA indicate their important regulatory roles in controlling the rate of hepatocyte migration and restitution following the creation of excisional wounds. Supported by the Medical Research Service of the Veterans Affairs. Presented at the Thirty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, San Francisco, Calif., May 19–22, 1996.     

Lidocaine Inhibits Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor and Suppresses Proliferation of Corneal Epithelial Cells

Background: Although lidocaine is recognized as an excellent topical corneal analgesic, its toxic effect on corneal epithelial cells limits its use during corneal epithelial wound healing. Mechanism of the impairment of corneal reepithelialization with lidocaine, however, has not been evaluated. The authors' previous study revealed that lidocaine inhibits the activity of tyrosine kinase receptors through the interaction with specific amino acid sequences around autophosphorylation sites, including acidic, basic, and aromatic amino acids. Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with an important role in epithelial cell proliferation after corneal wounding, also possesses these amino acids sequences around autophosphorylation sites. The authors hypothesized that lidocaine would suppress tyrosine kinase activity of EGFR and would impair corneal epithelial cell proliferation.

Methods: To investigate the effect of lidocaine (4 [mu]M-40 mM) on epidermal growth factor (EGF)-stimulated autophosphorylation of EGFR, the authors studied purified EGFR in microtubes. They cultured human corneal epithelial cells (HCECs) with EGF and lidocaine to investigate the effect of lidocaine on cell proliferation and on autophosphorylation of EGFR in HCECs.

Results: Lidocaine (>= 400 [mu]M) significantly suppressed EGF-stimulated autophosphorylation of the purified EGFR. In the HCEC study, EGF alone stimulated cell proliferation and increased autophosphorylation of EGFR in HCECs. Lidocaine (>= 400 [mu]M) significantly suppressed both the proliferation of HCECs promoted by EGF and EGF-stimulated autophosphorylation of EGFR.