Epiregulin Research Articles

Involvement of peroxiredoxin 2 in cumulus expansion and oocyte maturation in mice.

Peroxiredoxin 2 (Prdx2), an antioxidant enzyme, is expressed in the ovary during the ovulatory process. The aim of the present study was to examine the physiological role of Prdx2 during ovulation using Prdx2-knockout mice and mouse cumulus-oocyte complex (COC) from WT mice. Two days of treatment of immature mice (21-23 days old) with equine chorionic gonadotrophin and followed by treatment with human chorionic gonadotrophin greatly impaired cumulus expansion and oocyte maturation in Prdx2-knockout but not wild-type mice. Treatment of COCs in culture with conoidin A (50µM), a 2-cys Prdx inhibitor, abolished epiregulin (EPI)-induced cumulus expansion. Conoidin A treatment also inhibited EPI-stimulated signal molecules, including signal transducer and activator of transcription-3, AKT and mitogen-activated protein kinase 1/2. Conoidin A treatment also reduced the gene expression of EPI-stimulated expansion-inducing factors (hyaluronan synthase 2 (Has2), pentraxin 3 (Ptx3), TNF-α induced protein 6 (Tnfaip6) and prostaglandin-endoperoxide synthase 2 (Ptgs2)) and oocyte-derived factors (growth differentiation factor 9 (Gdf9) and bone morphogenetic protein 15 (Bmp15)). Furthermore, conoidin A inhibited EPI-induced oocyte maturation and the activity of connexins 43 and 37. Together, these results demonstrate that Prdx2 plays a role in regulating cumulus expansion and oocyte maturation during the ovulatory process in mice, probably by modulating epidermal growth factor receptor signalling.

FimH-based display of functional eukaryotic proteins on bacteria surfaces

The demand for recombinant proteins for analytic and therapeutic purposes is increasing; however, most currently used bacterial production systems accumulate the recombinant proteins in the intracellular space, which requires denaturating procedures for harvesting and functional testing. We here present a novel FimH-based expression system that enables display of fully functional eukaryotic proteins while preventing technical difficulties in translocating, folding, stabilizing and isolating the displayed proteins. As examples, Gaussia Luciferase (GLuc), epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and epiregulin (EPRG) were expressed as FimH fusion proteins on the surface of E. coli bacteria. The fusion proteins were functionally active and could be released from the bacterial surface by specific proteolytic cleavage into the culture supernatant allowing harvesting of the produced proteins. EGFR ligands, produced as FimH fusion proteins and released by proteolytic cleavage, bound to the EGF receptor (EGFR) on cancer cells inducing EGFR phosphorylation. In another application of the technology, GLuc-FimH expressed on the surface of bacteria was used to track tumor-infiltrating bacteria by bioluminescence imaging upon application to mice, thereby visualizing the colonization of transplanted tumors. The examples indicate that the FimH-fusion protein technology can be used in various applications that require functionally active proteins to be displayed on bacterial surfaces or released into the culture supernatant.

Initiation of prolyl cis-trans isomerisation in the CDR-H3 loop of an antibody in response to antigen binding

Proline cis-trans isomerisation is a regulatory mechanism used in a range of biological processes, and is related to various diseases such as Alzheimers disease and cancer. However, the details of the exact molecular mechanism by which it occurs are not known. Using X-ray crystallography, proline isomerisation has been shown to occur following formation of an antigen-antibody complex between the target epiregulin (EPR) and the antibody 9E5, at proline (Pro103), located in the third complementarity-determining region (CDR) of the heavy chain of 9E5. To obtain an accurate description of the pathway involved in cis-trans isomerisation in this system, we performed ten independent long molecular dynamics (MD) simulations starting at a stable transient bound structure obtained from many short binding MD simulations. As a result, we were able to describe the process by which cis-trans isomerisation is initiated, and suggest a catalysis mechanism for cis-trans isomerization in this antigen-antibody system. We found that Asp102, which is immediately adjacent to Pro103, rotates while changing its interacting partner residues in the light chain of 9E5, and at the same time EPR polar residues help to stabilise the intermediate states in the isomerisation process by interacting strongly with Asp102.

Conformational Changes in Antigen-Antibody Binding: Molecular Dynamics Study

Proline cis-trans isomerization have emerged as an effective regulatory mechanism in a wide range of biological processes related to Alzheimer disease and cancer. However, the details of the mechanism still remain to be clarified. The cis-trans isomerization has been observed in an antigen-antibody system, epiregulin (EPR) and its antibody by X-ray crystallography. Anti EPR antibody, 9E5 has a proline at the residue 103 in the third complementarity-determining region of the heavy chain (CDR-H3), which is in the cis conformation for EPR free 9E5 (apo 9E5) and the trans one for the complex of EPR-9E5 obtained from X-ray crystallography (we refer this complex as “trans-Complex”). Because the cis-trans isomerization is known to occur slowly, with the duration ranging from several minutes to hours, we investigated whether EPR forms a stable complex with apo 9E5 before the isomerization occurs. To identify a stable EPR-binding structure with apo 9E5, we conducted extensive binding molecular dynamics (MD) simulations and additional long MD simulations. An EPR-9E5 complex of which the bound structure is highly similar to that of the trans-Complex was found. We refer this complex as “cis-Complex”. We then investigated the structural stability and conformational changes of the cis-Complex. The cis-Complex is stable for at least 4 μs. It is found that the CDR-H3 loop plays important role for the EPR binding process in which the CDR-H3 loop interacts with EPR strongly. The interaction energy between the EPR and 9E5 is lower than that for the trans-Complex by more than 100 kJ/mol. From 10 μs-MD simulations of the cis-Complex, it is also found that the slight conformal changes of the Pro103 occur slowly during 10 μs.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro103. A molecular dynamics simulation and mutational analyses revealed that Arg40 in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Epidermal growth factor is a potential biomarker for poor cetuximab response in tongue cancer cells.

Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines. The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry. EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive. Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Type II cGMP-dependent protein kinase inhibits ligand-induced activation of EGFR in gastric cancer cells

Our previous data demonstrated that type II cGMP‑dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced MAPK/ERK/JNK‑mediated signal transduction through inhibiting the phosphorylation/activation of the epidermal growth factor receptor (EGFR). Since the EGFR also binds with several other ligands as well as EGF, the present study was designed to investigate whether PKG II inhibited transforming growth factor-α (TGF-α), betacellulin (BTC) and epiregulin (EPR) induced phosphorylation/activation of the EGFR and consequent MAPK/ERK‑mediated signaling. The human gastric cancer cell line AGS, was infected with adenoviral constructs encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and was treated with 8-pCPT-cGMP to activate the kinase. Western blotting was applied to detect the phosphorylation of EGFR and MAPK/ERK. The results demonstrated that treatment with EGF (100 ng/ml, 5 min), TGF-α (100 ng/ml, 5 min), BTC (100 ng/ml, 5 min) and EPR (100 ng/ml, 5 min) increased the tyrosine (tyr) 1068 phosphorylation of the EGFR and the threonine (thr) 202/tyr 204 phosphorylation of MAPK/ERK. Infecting the cells with Ad-PKG II and stimulating the kinase with 8-pCPT-cGMP efficiently inhibited the phosphorylation of the EGFR and MAPK/ERK induced by EGF, TGF-α, BTC and EPR. The results indicated that PKG II also inhibits the activation of the EGFR caused by diverse ligands of the receptor.

Constant Domain of Fab Fragment Affects Antigen Binding of Antibodies: Molecular Dynamics Study

The fragment antigen binding (Fab) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain (Fv), which contain the heavy (H) and the light (L) chains. Antibodies bind antigens via contacts with amino acids in complementarity-determining regions (CDRs) in Fv domain. It may seem to be plausible that there is no difference of the binding structure and affinity between Fv and Fab format if the both format have same CDRs. Recently, however, numerous studies have compared the binding affinity of the single chain Fv (scFv) and Fab variants of the same antibody and have found that the changing the format to a Fab resulted in equal or higher binding affinity. However, the affects of the structural format on the antibody binding affinity remains poorly understood. In order to investigate the difference of physical properties between Fab and Fv fragments, from a structural and thermodynamical point of view, we have carried out 1 μs molecular dynamics (MD) simulations for each format. We have used the antibodies of epiregulin (EPR) as a simulation model, which is a member of the epidermal growth factor (EGF) family and a factor affecting pancreatic cancer, exhibiting high biological activities.We have found that constant region of Fab fragment of the antibody play an important role in the Fv domain stability, which reduce a fluctuation between H chain and L chain, and reduce the surface area of basic and hydrophobic amino acid exposed to solvent. And also find that binding free energy of Fab fragments and EPR is lower than that of Fv fragments. This indicates that the presence of the constant region of Fab fragment affects the binding structure of EPR and antibody.

Expression of the epidermal growth factor system in human middle ear cholesteatoma

Conclusion: The detection of the HER4 receptor in 50% of cholesteatomas but never in the reference tissue, and the increased expression of its activating ligand EPI, suggest that EPI-mediated activation of HER4 might play a role in cholesteatoma growth. Objective: To investigate the expression of the epidermal growth factor (EGF) system in human middle ear cholesteatoma. Methods: Forty-seven patients referred for surgery due to cholesteatoma were included in the study. Clinical data were collected. Biopsies of cholesteatoma and skin from the external ear canal were obtained during surgery. mRNA expression was quantified with real-time PCR. The corresponding proteins were visualized using immunohistochemistry. Results: A systematic investigation of all four receptors, HER1, HER2, HER3, and HER4, and the ligands EGF, transforming growth factor (TGF)-α, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin (EPI) of the EGF system is presented. At the mRNA level, the study demonstrates an up-regulation of mRNA encoding EPI and AR. In contrast HER1 and EGF were down-regulated. HER4 mRNA could be detected in 50% of cholesteatoma and 20% of reference tissues, and the HER4 protein was detectable only in cholesteatoma tissue. HER1 and HER2 were also visualized by immunohistochemistry, whereas the ligands EPI, AR, and EGF were undetectable with our methods.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

Molecular Dynamics Studies of Protein Targets for Cancer

In this meeting, we present the molecular dynamics studies of two protein targets for cancers. The one is epiregulin (EPR), which is a member of the epidermal growth factor family and a factor affecting pancreatic cancer. Recently, it was observed that the binding affinity of EPR without S-S bonds for an antibody is lower than that of EPR with S-S bonds, and that the affinity of a long form type (extracellular domain) of EPR without S-S bonds is higher than that of a mature type without S-S bonds. To investigate the effect of the S-S bonds on the structural stability of EPR, we have performed molecular dynamics (MD) simulation for these three types of EPR. From our simulations, it has found that the S-S bonds reduce the structural fluctuation of EPR, and the structure of the mature domain in the long form type is similar to the mature type with S-S bonds.The other protein target is the loop region between fibronectin type III domains of roundabout-1 that is a receptor for Slit1 and Slit2 in axon growth cones and differentially expressed in cancers. We have investigated the structural stability and thermodynamic property of the loop region using MD simulation. We have calculated two types of peptide of different size: the peptide composed of 26 amino acid residues corresponding to the full region of the fluctuating loop, and the shorter one of 20 amino acid residues. In the 20 amino acid peptide calculations, we have observed two different stable conformations, which are stable during more than 1.5 μs. We have found that for the one conformation, the intramolecular interaction energy contributes to the thermodynamical stability, while for the other one, water binding to the turn part of the peptide is dominant contribution.

Abstract 251: Kinome-wide RNAi screen identification of synthetic lethal interactions in BRAF-mutant melanoma

Abstract BACKGROUND. Mutations in the BRAF oncogene are prevalent in many types of solid cancers, where they promote cell proliferation and transformation by constitutively activating the RAF-MEK-ERK signaling pathway. Inhibitors of BRAF have been developed as potential cancer therapies but unfortunately many have performed poorly in clinical trials. As an alternative approach, we performed RNA interference (RNAi) screening to identify genes that are synthetically lethal with the BRAF-mutant oncogene in malignant melanoma cell lines. Recent advances in RNAi and deep sequencing technologies have made it possible to systematically interrogate the genome for synthetic lethal interactions. METHODS. Two BRAF-mutant melanoma cell lines (SKMEL28 and Malme3M) and one wild-type BRAF cell line (MeWo) were infected with a subset of The RNAi Consortium (TRC) shRNA library comprising all the kinases in the human genome as well as the genes involved in the tyrosine kinase>RAS>RAF>MEK>ERK pathway. Cells were collected at 4 and 19 days post-infection, genomic DNA was isolated, shRNAs were PCR amplified and subjected to massively parallel sequencing. Statistical analysis identified ∼200 shRNAs that were selectively depleted in BRAF-mutant but not wild-type BRAF cell lines; individual canidate shRNA were tested in a panel of 6 melanoma cell lines 3 BRAF-mutant (UACC62, UACC257, Malme3M) and 3 wild-type BRAF (WM3912, WM3918, MeWo). SUMMARY. The screen identified a diverse number of genes that are synthetically lethal with the BRAF-mutant oncogene in human malignant melanoma. Not surprisingly, a number of the identified synthetic lethal genes are known downstream effectors of BRAF or known scaffold proteins involved in MAPK signaling. Paradoxically, epiregulin (EPGN), a known ligand of the epidermal growth factor receptor (EGFR) and EGFR itself, which are upstream of the BRAF oncogene were also identified in the screen as synthetically lethal with mutant BRAF. CONCLUSIONS. The BRAF oncogene is synthetically lethal with genes involved in diverse cellular processes such as kinase signaling, cell division, nuclear shuttling and cell metabolism. Many of the genes identified in the RNAi screen are known effectors of the MAPK cascade. Paradoxically, two members of the MAPK cascade, EGFR and its ligand EPGN, both of which are upstream of BRAF, were identified in the screen and validated to be synthetically lethal with mutant BRAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 251. doi:10.1158/1538-7445.AM2011-251

Evolution of the predictive markers amphiregulin and epiregulin mRNAs during long-term cetuximab treatment of KRAS wild-type tumor cells

Molecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX's molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury

Epiregulin (EPI) and amphiregulin (AR) are epidermal growth factor receptor (EGFR) ligands implicated in mucosal repair and tumorigenesis. We have shown that Toll-like receptor 4 (TLR4) induces intestinal epithelial cell (IEC) proliferation by activating EGFR through AR expression. We examined whether TLR4 differentially regulates expression of EGFR ligands in response to mucosal injury. The human IEC line SW480 was examined expression of EGFR ligands, EGFR phosphorylation, and proliferation in response to lipopolysaccharide (LPS). Small-interfering RNA (siRNA) was used to block TLR4. Neutralizing antibodies to EGFR ligands were used to examine inhibition of LPS-dependent EGFR activation. Acute colitis and recovery were examined in the mice given 2.5% dextran sodium sulfate (DSS). Colonic secretion of EPI and AR was analyzed by enzyme-linked immunosorbent assay. LPS selectively induces EPI and AR but not other EGFR ligands. LPS induced early EPI mRNA expression between 30 min and 24 h. The neutralizing antibodies to EPI and AR prevented activation of EGFR by LPS. LPS induces IEC proliferation (200%, P=0.01) in 24 h but blocking EPI and AR significantly decreased proliferation. In vivo, mucosal EPI and AR expression are significantly decreased in TLR4−/− mice (P=0.02) compared to wild-type mice during acute colitis. EPI and AR exhibit different kinetics in response to mucosal damage: EPI expression is upregulated acutely at day 7 of DSS, but falls during recovery at day 14. By contrast, a sustained upregulation of AR expression is seen during mucosal injury and repair. We show that TLR4 regulates EPI and AR expression and that both these EGFR ligands are necessary for optimal proliferation of IEC. The diverse kinetics of EPI and AR expression suggest that they function in distinct roles with respect to acute injury vs repair. Our results highlight the role of bacterial sensing for IEC homeostasis and may lead to targeted therapy for mucosal healing and prevention of tumorigenesis.

Bradykinin Decreases Podocyte Permeability through ADAM17-Dependent Epidermal Growth Factor Receptor Activation and Zonula Occludens-1 Rearrangement

Recent data show that increases in bradykinin (BK) concentration contribute to the beneficial effects of angiotensin-converting enzyme inhibitor (ACEI) treatment in chronic kidney disease. However, the possible role of BK in attenuated proteinuria, often seen in ACEI-treated patients, is not well studied. Here, we report that BK decreases mouse podocyte permeability through rearrangement of the tight junction protein zonula occludens-1 (ZO-1) and identify some of the major signaling events leading to permeability change. We show that BK2 receptor (BK2R) stimulation transactivates the epidermal growth factor receptor (EGFR). EGFR transactivation is mediated by a disintegrin and metalloenzyme (ADAM) family members, which are required for both extracellular signal-regulated kinase (ERK) and EGFR activation by BK. Using a gene-silencing approach we observed that both BK-induced ERK activation and BK-induced permeability decrease in podocytes is attenuated by ADAM17 down-regulation, and we identified epiregulin (ER) as the EGFR ligand participating in ADAM-dependent BK2R-EGFR cross-talk. EGFR inhibition attenuated both ZO-1 rearrangement and BK-induced permeability decreases in podocyte. We propose that ZO-1 redistribution is an important element of BK-induced permeability change and the signaling events involved in ZO-1 rearrangement include transactivation of the EGFR via ADAM17 activation and ER shedding. Our data indicate that ADAM17 and the EGFR may be potential novel therapeutic targets in diabetic nephropathy and other chronic kidney diseases.

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.

Mechanical stretch promotes fetal type II epithelial cell differentiation via shedding of HB‐EGF and TGF‐α

The mechanisms by which mechanical forces promote fetal lung development are not fully understood. Here, we investigated differentiation of fetal type II epithelial cells via the epidermal growth factor receptor (EGFR) in response to mechanical strain. First, we showed that incubation of embryonic day (E) 19 fetal type II cells with recombinant heparin-binding EGF-like growth factor (HB-EGF) or transforming growth factor (TGF)-alpha, but not with amphiregulin (AR), betacellulin (BTC) or epiregulin (EPR), increased fetal type II cell differentiation, as measured by surfactant protein B/C mRNA and protein levels. Next, we demonstrated that 5% cyclic stretch of E19 monolayers transfected with plasmid encoding alkaline phosphatase (AP)-tagged ligands shed mature HB-EGF and TGF-alpha into the supernatant and promoted type II cell differentiation. Release of these ligands was also observed in E19 cells subjected to higher degrees of cyclic strain, but not in cells exposed to continuous stretch. Interestingly, the addition of fibroblasts to type II cell cultures did not enhance release of HB-EGF. Whereas HB-EGF shedding was also detected in E18 cells exposed to 5% cyclic stretch, release of this ligand after 2.5% sustained stretch was restricted to cells isolated on E18 of gestation. In addition, mechanical stretch released EGF, AR and BTC. We conclude that mechanical stretch promotes fetal type II cell differentiation via ectodomain shedding of HB-EGF and TGF-alpha. The magnitude of shedding varied depending on gestational age, ligand, and strain protocol. These studies provide novel mechanistic information potentially relevant to fetal lung development and to mechanical ventilation-induced lung injury.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

042 Role of Erbb3 and Epidermal Growth Factor-Like Ligands on Wound Healing

The role of the EGF superfamily of growth factors and their receptors during wound healing continues to unfold. The aim of the study was to determine the effect of overexpression of erbB3 in wound healing and whether topical application of its various ligands enhances repair. Partial thickness porcine wounds were transfected with recombinant adenoviral particles containing erbB3 receptor gene or a vehicle β-galactosidase gene (Lac Z). Highly similar ligands: Epidermal growth factor (EGF), Epiregulin (EPR), Heparin-binding epidermal growth factor (HB-EGF), Heregulin (HER) were topically applied to wounds boosted with erbB3 receptors. Enhanced wound healing parameters were assessed 5 days after initial treatment for reepithelization, dermal depth, macrophage infiltration, proliferating cells, angiogenesis, and apoptotic cells. Treatment with EPR and HB-EGF promoted greater resurfacing of the epidermal layer than EGF, HER, erbB3 only or vehicle treated wounds. Neodermal formation as measured by dermal or granulation depth was significantly different in the EPR and HB-EGF treated wounds compared to EGF, HER, erbB3 only or vehicle. Numerous hallmarks of enhanced wound maturity (fewer infiltrating macrophages, significant reductions in neovascularization, fibroblastic proliferation) were noted in EPR- and HB-EGF-treated wounds transfected with erbB3 as compared to vehicle controls. Only erbB3 wounds treated with these particular ligands showed significant increases in apoptotic cells. Our results indicate that wounds treated with this erbB3 receptor form supplied with topical application of EPR and HB-EGF exhibit synergistic and accelerating reparative effects on porcine partial thickness wounds as compared to vehicle. These in vivo findings suggest the complexity and evidence of synergism between the erbB receptor and its various ligands on wound healing. (This research work was funded by the NIH GM40437.)

The Kinetics of the Hydrogen/Deuterium Exchange of Epidermal Growth Factor Receptor Ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 310-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20min−1 (Tyr), 0.09min−1 (Arg, β-turns), and 1.88×10−3min−1 (β-sheets and 310-helix); and for TGF-α 0.91min−1 (Tyr), 0.27min−1 (Arg, β-turns), and 1.41×10−4min−1 (β-sheets). The time constants for AR 0.47min−1 (Tyr), 0.04min−1 (Arg), and 1.00×10−4min−1 (buried 310-helix, β-turns, and β-sheets); for HB-EGF 0.89min−1 (Tyr), 0.14min−1 (Arg and 310-helix), and 1.00×10−3min−1 (buried 310-helix, β-sheets, and β-turns); and for epiregulin 0.16min−1 (Tyr), 0.03min−1 (Arg), and 1.00×10−4min−1 (310-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.