Member Of Epidermal Growth Factor Family Research Articles

Transcription factor Dlx2 protects from TGFβ-induced cell-cycle arrest and apoptosis

Acquiring resistance against transforming growth factor β (TGFβ)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFβ's tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFβ-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFβ receptor II (TGFβRII) gene expression and reduces canonical, Smad-dependent TGFβ signalling and expression of the cell-cycle inhibitor p21(CIP1) and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFβ from its tumour suppressive to its tumour-promoting functions.

The role of neuregulin4 and HER4 in gastrointestinal malignant lymphoma

The human epidermal growth factor (EGF) receptor (HER) family consists of four receptors that bind to ligands sharing an EGF-like motif. The HER family of receptor tyrosine kinases and their ligands (EGF family) are known to play a significant role in gastrointestinal cancer. In particular, the EGF receptor, HER1, is one of the main candidates for the molecular-targeted therapy of colon cancer, and HER2 is a candidate for the treatment of gastric cancer which overexpresses HER2. In contrast, the role of the HER and EGF families in malignant lymphoma has not been fully elucidated. In this study, we investigated the expression and function of the HER and EGF families in lymphoma cell lines and tumor samples. Reverse transcription polymerase chain reaction revealed that the ligands for HER1 were mainly expressed in gastric cancer and colon cancer cell lines, but not in lymphoma cell lines. On the other hand, the EGF family member, neuregulin (NRG) 4, was highly expressed in lymphoma cell lines. Immunohistochemical analyses of malignant lymphoma clinical samples revealed that NRG4 and HER4 were mainly expressed in mucosa-associated lymphoid tissue (MALT) and follicular lymphoma. Immunoprecipitation of Raji and Daudi cell lines revealed that recombinant NRG4 induced the tyrosine phosphorylation of HER4. Additionally, recombinant NRG4 activated the proliferation of lymphoma cell lines. These findings suggest that the NRG4-HER4 axis plays a major role in the proliferation of malignant lymphoma cells in the gastrointestinal tract.

Neuregulin1 as Novel Therapy for Heart Failure

Neuregulin1 proteins (NRG1s) are epidermal growth factor (EGF) family members which are ligands for the ErbB receptor tyrosine kinases (RTKs). A decade of research has revealed that the NRG1-ErbB signaling is essential for the cardiac development and pivotal for maintaining the physiological function of the adult heart. The first evidence regarding the protective effect of the ErbB2 signaling in the adult heart came from clinical trials in breast cancer patients using Trastuzumab, a monoclonal antibody that blocks the ErbB2 receptor. The incidence of the New York Heart Association (NYHA) class III/IV heart failure increased five-fold in patients treated concurrently with chemotherapy drug doxorubicin and Trastuzumab compared to those treated with doxorubicin alone. Subsequent studies further show that stimulation of the ErbB2 signaling by NRG1s improves cardiomyocyte survival, growth and proliferation, maintains cardiac myofibril structure, counterbalances excessive β-adrenergic signaling and promotes angiogenesis in the heart. Injections of recombinant NRG1s improve cardiac function in animal models with myocardial infarction, doxorubicin, viral infection or pacing-induced heart failure. Recent clinical trials show that NRG1s are effective for improving the cardiac function in heart failure patients. These results suggest that NRG1s may become a new drug for the treatment of heart failure. NRG1s stimulate RTKs. This is different from Beta-blockers, ACE inhibitors (Angiotensin-Converting Enzyme) and Angiotensin II receptor blockers which inhibit the excessive activation of G-protein coupled receptors (GPCRs). A clear understanding of how NRG1-ErbB signaling regulates cardiac function is essential for successful use of NRG1s for heart failure. Here, we review the current knowledge of the NRG1-ErbB signaling in the heart and discuss the potential use of NRG1s as novel therapy for heart failure.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Role of guanine‐binding protein inhibitory alpha in activation of the Akt/mTORC1 pathway by growth factors

The Akt/mTOR pathway is essential for cell growth, migration and survival mediated by growth factors. Although it has been known that growth factor receptors called receptor tyrosine kinases (RTKs) and adaptor proteins including Grb2, Gabs, IRSs as well as FRSs are critical for activation of this pathway, it is still not well understood how these adaptor proteins are activated. We have found that the heterotrimeric guanine‐nucleotide binding protein (G protein) alpha inhibitory (Gia) is critical for mediating this activation. In Gi1a and Gi3a double knockout mouse embryonic fibroblasts (Gia1/3 DKO MEFs), the activation of the Akt/mTORC pathway was largely impaired by epidermal growth factor (EGF), EGF family members, fibroblast growth factors, and low doses of platelet derived growth factor (PDGF). In contrast, Gia1 and Gi3a double deficiency had no profound defect in activation of this pathway by insulin and insulin‐like growth factors. Mechanistic study results indicated that Gia proteins are important for the association of adaptor proteins and PI3K regulatory subunit p85. Further analysis revealed that loss of Gia proteins impaired MEFs migration and proliferation in response to growth factors. In addition, Gia proteins are important for cancer cell growth and migration. Overall, our results suggest that Gia proteins are new players in growth factor signaling and the pathogenesis process of cancer.

In vitro maturation of human germinal vesicle-stage oocytes: role of epidermal growth factor-like growth factors in the culture medium

In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side effects of gonadotrophin stimulation for IVF. The pregnancy rates from oocytes matured in vitro are still lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Recently, it was demonstrated that LH exerts its action on ovulation, at least in part, through stimulation of the production of the epidermal growth factor family members amphiregulin (Areg) and epiregulin (Ereg) in pre-ovulatory follicles, and they, in turn, serve as paracrine mediators of LH. We aimed to investigate the effect of supplementation of the medium with Areg and Ereg on the maturation rate of immature oocytes. A total of 105 sibling human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation were cultured in a complex defined medium either with or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/ml) for 24 h. Significantly more oocytes reached the metaphase II stage at 24 h in media supplemented with Areg and Ereg (75.5 versus 36.5%, P < 0.001). In vitro matured oocytes retrieved from the two subgroups had no statistically significant difference in fertilization and cleavage rates or morphology scores. Overall, a significantly higher number of Day 2 (52.8 versus 26.9% P < 0.01) and Day 3 (45.2 versus 23%, P < 0.05) embryos originated from GV oocytes cultured in the Areg- and Ereg-enriched medium. Supplementation of the maturation medium with Areg and Ereg improves the maturation of human GV oocytes in vitro.

Amphiregulin stimulates cumulus-oocyte expansion and oocyte maturation in rhesus macaques

OBJECTIVE: To determine the role of the epidermal growth factor (EGF) family member, amphiregulin (AREG), on rhesus macaque cumulus-oocyte expansion (C-OE) and oocyte maturation. DESIGN: In vitro study using cumulus-oocyte complexes (COCs) obtained from rhesus macaques. MATERIALS AND METHODS: COCs (n= 55) were incubated for 30 h in TALP media under the following conditions: media with monkey serum (MS, control) alone or with AREG (10 ng/ml or 50 ng/ml). A fluorescence-based technique was used to evaluate extracellular levels of hyaluronic acid (HA) in treated COCs, as its synthesis is critical for C-OE. COCs from healthy small antral follicles were isolated from the macaque ovarian medulla after collagenase treatment. Individual COCs (n=90) were cultured for 48 h with MS and gonadotropins alone or with AREG (10 ng/ml or 100 ng/ml). At 48h, germinal vesicle (GV) breakdown (metaphase I, MI), the presence of a polar body (metaphase II, MII), and spindle assembly was determined by microscopy. RESULTS: AREG treatment of rhesus macaque COCs induced HA levels in the matrix that surrounds the cumulus cells. In the absence of AREG, however, COC HA staining was minimal. AREG (10 ng/ml) incubation with rhesus macaque COCs increased the percentage of oocytes that progressed to the MII stage of meiosis relative to the control group (82% vs. 33%, p<0.05), as well as decreased the percentage of MI oocytes (6% vs. 39%, p<0.05). Surprisingly, the percentage of either MI or MII oocytes was not different between the 100 ng/ml AREG group and controls. CONCLUSION: These data indicate, for the first time in primates, that AREG may play a role in C-OE as well as in oocyte nuclear maturation at a specific concentration. Thus, further studies are warranted to determine if AREG can serve as an intrafollicular marker of C-OE and oocyte competence in primates.

Expression of epidermal growth factors and apoptosis markers in pancreatic ductal adenocarcinoma.

Epidermal growth factor family members: EGF, EGFR and the c-erbB-2(HER-2/neu) are involved in the growth of pancreatic ductal carcinoma, its invasiveness and metastases. Similarly, proteins regulating apoptosis can influence the development of pancreatic cancer. The aim of our study was to assess the expressions of EGF, EGFR, c-erbB-2, Bax and Bcl-xL in comparison with anatomo-clinical parameters. We also analyzed the relationship between the epidermal growth factors and apoptosis-regulating proteins. The levels of these proteins were determined immunohistochemically in 29 pancreatic ductal carcinoma cases. We found no correlation of EGF, EGFR, c-erbB-2, Bax and Bcl-xL with age and gender of patients, or histological type and grade of malignancy (G). However, we observed a very strong correlation between EGF, EGFR, Bax, Bcl-xL and lymph node metastases (p=0.000, p=0.001, p=0.008, p=0.012, respectively) and between EGF, EGFR and distant metastases (p=0.002, p=0.008, respectively). Moreover, we found a correlation between Bcl-xL and c-erbB-2 (p=0.030) and between EGF and Bax (p=0.041). These investigations seem to suggest that both epidermal growth factors (EGF, EGFR) and apoptosis-regulating proteins (Bax and Bcl-xL) play an essential role in lymph node involvement. Moreover EGF and EGFR are involved in distant metastases. The apoptosis markers appear to cooperate with epidermal growth factor proteins in the process of tumor spread.

Potential Involvement of Twist2 and Erk in the Regulation of Osteoblastogenesis by HB-EGF-EGFR Signaling

Epidermal growth factor (EGF) family members play important roles in the skeletal system. In this study, we examined the role of EGF receptor (EGFR) signaling in osteoblastogenesis in vitro. The expression of HB-EGF and epiregulin (EPR) was transiently induced within 24 h after osteogenic stimulation, but when preosteoblastic MC3T3-E1 cells were incubated with HB-EGF or EPR, osteoblast differentiation was inhibited. These effects were Ras-dependent, and ERK modulated Runx2 activity through the localization of Smad1 and the induction of Twist2. PI3-kinase was also required for the induction of Twist2. However, the inhibition of individual signaling pathways was not sufficient to overcome HB-EGF-mediated inhibition of osteoblast differentiation. Additionally, HB-EGF treatment promoted the proliferation of preosteoblasts, and this was associated with the downregulation of p27 at the protein level. These results suggest that HB-EGF-EGFR signaling inhibits the differentiation of osteoblasts by suppression of Runx2 transcriptional activity and enhances proliferation of preosteoblasts by downregulation of expression of p27.

Spatial localization of EGF family ligands and receptors in the zebrafish ovarian follicle and their expression profiles during folliculogenesis

The roles of epidermal growth factor (EGF) family in the ovary have received increasing attention recently. Despite this, the production sites of EGF family members in the ovarian follicle still remain controversial. Using zebrafish as the model, the present study investigated spatial distribution of several EGF family ligands and receptors in the follicle as well as their temporal expression profiles during folliculogenesis. RT-PCR analysis on the somatic follicle layer and oocyte revealed that all EGF family ligands examined ( egf, tgfa, btc and hbegf) were mostly or exclusively expressed in the oocyte. In contrast, their common receptor ( egfr) was expressed exclusively in the follicle layer. By comparison, members of activin family showed an opposite pattern of distribution. Activin subunits ( inhbaa and inhbb) were both expressed exclusively in the follicle layer whereas activin receptors and follistatin were abundantly present in the oocyte. During folliculogenesis, egf, tgfa and hbegf increased their expression together with egfr in the fast secondary growth phase. The developmental profiles of EGF family during embryogenesis appeared to argue for an important role for EGF family in folliculogenesis rather than embryogenesis as maternal molecules. The present study provided clear evidence for the existence of two paracrine pathways in the follicle, the oocyte-derived EGF family ligands and follicle cell-derived activins, which may mediate oocyte-to-follicle cell and follicle cell-to-oocyte communications, respectively. The functional relationship between these two signaling systems in the follicle is suggested by the observation that all four EGFR ligands examined significantly stimulated activin subunit expression in cultured follicle cells.

Astrocytes Reverted to a Neural Progenitor-like State with Transforming Growth Factor Alpha Are Sensitized to Cancerous Transformation

Gliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress.

Prolactin–Growth Factor Crosstalk Reduces Mammary Estrogen Responsiveness Despite Elevated ERα Expression

Most breast cancers that occur in women express estrogen receptor alpha (ERalpha). However, a large subset of these cancers either does not initially respond to anti-estrogen therapy or develops resistance to such treatment modalities. One postulated mechanism of this failure is signaling cross talk between hormones and local growth factors. To examine these complex interactions in vivo, we assessed the effects of estrogen on transforming growth factor alpha (TGFalpha)- and prolactin (PRL)-induced mammary tumorigenesis in transgenic mice. Both PRL and estrogen reduced the latency of TGFalpha-induced oncogenesis, resulting in tumors that were variably ERalpha-positive, but were progesterone receptor-negative. However, despite elevated ERalpha levels in NRL-PRL/TGFalpha glands, tumor latency was not reduced with increasing estrogen levels, nor increased after ovariectomy. Furthermore, PRL and TGFalpha in combination blocked the mitogenic effects of estrogen, dramatically reduced progesterone receptor levels, and diminished ERalpha down-regulation in response to circulating estrogen levels, in contrast to the other genotypes. Notably, however, ductal morphology remained responsive to estrogen, indicating that TGFalpha and PRL in combination can inhibit some, but not all, estrogenic signals. Both in vitro and in vivo, PRL and TGFalpha cooperatively enhanced Akt phosphorylation, which is associated with endocrine resistance in human disease. These findings provide insight into the interactions of PRL with growth factors during mammary oncogenesis and suggest combinatorial approaches that may result in improved therapeutic efficacy.

Transcriptional Activity of Human Epidermal Growth Factor Receptor Family and Angiogenesis Effectors in Locoregionally Recurrent Head and Neck Squamous Cell Carcinoma and Correlation with Patient Outcome

Locoregional recurrence is the most common failure pattern in patients with head and neck squamous cell carcinoma (HNSCC). We retrospectively identified 41 HNSCC patients with locoregional relapse and used kinetic reverse transcription-polymerase chain reaction (kRT-PCR) in order to study fresh-frozen tumour messenger RNA (mRNA) levels of the Human Epidermal growth factor family members HER1-4, the Vascular Endothelial Growth Factors (VEGFs) A, B, C, D, and their receptors VEGFR1, 2, 3. High VEGF-C and VEGFR3 tumour mRNA expression correlated with relapse beyond the primary locus (neck nodes or soft tissues, P < .05). Tumours with regional nodal involvement at diagnosis more often exhibited high transcriptional activity of VEGFR1 and VEGFR3 at the time of relapse (P < .05). At a median follow-up of 52 months from the time of locoregional recurrence, patients with high VEGF-C tumours at relapse had significantly poorer postrelapse progression-free survival (R-PFS, 5 versus 47 months, log-rank P = .052) and a trend for inferior postrelapse overall survival (R-OS, 22 versus 44 months, log-rank P = .076) in comparison to low VEGF-C tumours. Similar association with dismal outcome was seen for its receptor, VEGFR3 tumoural mRNA levels (log-rank P = .060). In contrast, suppressed tumour transcription of VEGF-D was associated with poorer post-relapse survival, though statistical significance was not reached. Active transcription of the VEGF-C/VEGFR3 axis in recurrent HNSCC is associated with failure at neck soft tissues/lymph nodes and inferior survival post-relapse.

Differential regulation of betacellulin and heparin-binding EGF-like growth factor in cultured zebrafish ovarian follicle cells by EGF family ligands

Recently the roles of epidermal growth factor (EGF) family ligands in vertebrate ovaries have received increasing attention, including betacellulin (BTC), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor α (TGFα), epiregulin, and EGF itself. In the zebrafish ( Danio rerio), four members of EGF family have been identified by either molecular cloning or genome sequencing, which are EGF, TGFα, BTC, and HB-EGF. Although they are mostly expressed in the oocytes in the ovary, the present study demonstrated the expression of all the four EGF family ligands ( egf, btc, tgfa, and hbegf) in cultured zebrafish follicle cells albeit at very low levels. Treatment of the cultured follicle cells with EGF, BTC, and HB-EGF demonstrated differential effects of these ligands on the expression of themselves. While the expression of egf was rather non-responsive to EGF, BTC, and HB-EGF, the expression of btc was consistently down-regulated by all the three molecules. In contrast, hbegf increased its expression in response to these molecules. These results suggest that there is an EGF signaling network in the zebrafish ovarian follicle, and the functionality of this network is self-regulated by its own members.

The Cytoplasmic Domain of proEGF Negatively Regulates Motility and Elastinolytic Activity in Thyroid Carcinoma Cells

The intracellular domains of the membrane-anchoring regions of some precursors of epidermal growth factor (EGF) family members have intrinsic biologic activities. We have determined the role of the human proEGF cytoplasmic domain (proEGFcyt) as part of the proEGF transmembrane-anchored region (proEGFctF) in the regulation of motility and elastinolytic invasion in human thyroid cancer cells. We found proEGFctF to act as a negative regulator of motility and elastin matrix penetration and the presence of proEGFcyt or proEGF22.23 resulted in a similar reduction in motility and elastinolytic migration. This activity was counteracted by EGF-induced activation of EGF receptor signaling. Decreased elastinolytic migratory activity in the presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with decreased secretion of elastinolytic procathepsin L. The presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with the specific transcriptional up-regulation of t-SNARE member SNAP25. Treatment with siRNA-SNAP25 resulted in motility and elastin migration being restored to normal levels. Epidermal growth factor treatment down-regulated SNAP25 protein by activating EGF receptor-mediated proteasomal degradation of SNAP25. These data provide first evidence for an important function of the cytoplasmic domain of the human proEGF transmembrane region as a novel suppressor of motility and cathepsin L-mediated elastinolytic invasion in human thyroid carcinoma cells and suggest important clinical implications for EGF-expressing tumors.

Postsynaptic expression of an epidermal growth factor receptor regulates cholinergic synapse formation between identified molluscan neurons

Epidermal growth factor (EGF) family members are conserved in both vertebrates and invertebrates. Recent studies suggest that EGF ligands in invertebrates may have neurotrophic actions that possibly compensate for the apparent absence of neurotrophins in these species. In this study, we have cloned an EGF receptor from the mollusk Lymnaea stagnalis (L-EGFR), and shown that L-EGFR is the receptor for a previously identified EGF-like peptide in Lymnaea, named Lymnaea EGF (L-EGF). Knock-down of L-EGFR expression prevented L-EGF-induced excitatory synapse formation between identified cholinergic neuron visceral dorsal 4 (VD4) and its postsynaptic partner left pedal dorsal 1 (LPeD1). Moreover, knock-down of L-EGFR also prevented synapse formation induced by Lymnaea brain conditioned medium, suggesting that L-EGF is the most important, if not the only, brain-derived factor that promotes excitatory cholinergic synapse formation in Lymnaea. Thus, our data establish canonical EGF/EGFR signaling as an important synaptotrophic mechanism in invertebrates.

The Effect of Interleukin-4 and Amphiregulin on the Proliferation of Human Airway Smooth Muscle Cells and Cytokine Release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)-BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients

BackgroundThe epidermal growth factor (EGF) and EGF receptor (EGFR) families play important roles in the hyperplastic growth of several tissues as well as tumor growth. Since synovial hyperplasia in rheumatoid arthritis (RA) resembles a tumor, involvement of the EGF/EGFR families in RA pathology has been implied. Although several reports have suggested that ErbB2 is the most important member of the EGFR family for the synovitis in RA, it remains unclear which members of the EGF family are involved. To clarify the EGF-like growth factors involved in the pathology of RA, we investigated the expression levels of seven major EGF-like growth factors in RA patients compared with those in osteoarthritis (OA) patients and healthy control subjects.MethodsThe expression levels of seven EGF-like growth factors and four EGFR-like receptors were measured in mononuclear cells isolated from bone marrow and venous blood, as well as in synovial tissues, using quantitative RT-PCR. Further evidence of gene expression was obtained by ELISAs. The proinflammatory roles were assessed by the growth-promoting and cytokine-inducing effects of the corresponding recombinant proteins on cultured fibroblast-like synoviocytes (FLS).ResultsAmong the seven EGF-like ligands examined, only amphiregulin (AREG) was expressed at higher levels in all three RA tissues tested compared with the levels in OA tissues. The AREG protein concentration in RA synovial fluid was also higher than that in OA synovial fluid. Furthermore, recombinant human AREG stimulated FLS to proliferate and produce several proinflammatory cytokines, including angiogenic cytokines such as interleukin-8 and vascular endothelial growth factor (VEGF), in a dose-dependent manner. The VEGF mRNA levels in RA synovia and VEGF protein concentrations in RA synovial fluid were significantly higher than those in the corresponding OA samples and highly correlated with the levels of AREG.ConclusionThe present findings suggest that AREG functions to stimulate synovial cells and that elevated levels of AREG may be involved in the pathogenesis of RA.

Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation

The amount of mucin on the ocular surface is regulated by the rate of mucin synthesis, mucin secretion, and the number of goblet cells. We have previously shown that cholinergic agonists are potent stimuli of mucin secretion. In contrast, there have been no studies on the control of goblet cell proliferation. In this study we investigate the presence of the EGF family of growth factors and their receptors in rat conjunctiva and cultured rat conjunctival goblet cells as well as their effects on activation of signaling intermediates and goblet cell proliferation. Rat conjunctival goblet cells were grown in organ culture and identified as goblet cells by their morphology and positive staining for the lectin UEA-1 and cytokeratin 7. In the rat conjunctiva, the presence of the EGF family members epidermal growth factor (EGF), transforming growth factor α (TGF-α), heparin binding EGF (HB-EGF), and heregulin was determined by RT-PCR. The receptors for these ligands, EGF receptor (EGFR), erbB2, erbB3, and erbB4 were detected in both rat conjunctiva and goblet cells by Western blot analysis. Immunofluorescence microscopy of conjunctival tissue determined that EGFR was present as punctate staining in the cytoplasm of conjunctival goblet cells while ErbB2 was present in the basolateral and lateral membranes of goblet cells. ErbB3 was localized to the cytosol of rat conjunctival goblet cells. In cultured goblet cells, EGFR and ErbB2 were present in the perinuclear area of the cells. ErbB3 was widely distributed throughout the cytoplasm of the cells. ErbB4 was not detected in either the conjunctiva or goblet cells by immunofluorescence microscopy. Using a multiplex assay system we measured phosphorylation (activation) of p44/p42 mitogen-activated protein kinase (MAPK), also known as ERK, Jun N-terminal kinase (JNK), p38 MAPK and AKT (also known as protein kinase B), molecules known to be activated by EGF receptor members. EGF, TGF-α and HB-EGF activated the signaling intermediate proteins whereas heregulin did not. No EGF family member significantly activated AKT. Consistent with these findings, EGF, TGF-α and HB-EGF each stimulated goblet cell proliferation as measured by WST-1 assay or immunofluorescence microscopy using an antibody against Ki-67, a protein expressed in dividing cells. Heregulin did not cause goblet cell proliferation. We conclude that multiple members of the EGF family, EGF, TGF-α and HB-EGF, and heregulin are present with three of the four erbB receptor subtypes. EGF, TGF-α and HB-EGF all stimulated the activation of signaling intermediates and caused goblet cell proliferation.

Role of epidermal growth factor and its receptor family in ocular surface wound healing

The epidermal growth factor (EGF) and its receptor (ErbB) family have been shown to be involved in cell differentiation, proliferation and migration. EGF family members are expressed in the tear constantly and ErbB is expressed in ocular surface tissues. EGFR activation and subsequent intracellular signaling are required for corneal wound healing. They are important for the maintenance of the integrity of the ocular surface. The purpose of this review was to provide an overview of the role of EGF in the healing process of corneal epithelial cells after trauma.