Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn(2+)-induced EGFR activation. Exposure to Zn(2+) induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn(2+) on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn(2+)-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn(2+)-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn(2+)-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn(2+)-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn(2+) induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.

Impaired wound healing in diabetic patients is associated with altered inflammatory responses, poor angiogenesis, deficient extracellular matrix (ECM) component, and peripheral neuropathy. To develop a wound dressing that is capable of the controlled delivery of bioactive small molecules that can improve diabetic wound healing, dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) (PCL) fiber (PCLF/DMOG) meshes are fabricated by electrospinning, and the effects of the PCLF/DMOG meshes on wound healing in diabetic rats are evaluated. Electrospun PCLF/DMOG meshes increase not only the wound closure, re-epithelialization ratio, epithelial maturation (K-10-positive epidermis), and collagen-positive area but also the numbers of angiogenic marker (CD-31)-positive and neuronal marker (neurofilament)-positive cells compared to PCLF (p < 0.05). In in vitro examinations, RAW264.7 macrophages grown on PCLF/DMOG meshes enhance the expression of growth factors (IGF-1, HB-EGF, and NGF) and anti-inflammatory factors (TGF-β1 and IL-4) but decrease that of pro-inflammatory factors (IL-1β and IL-6). Keratinocyte migration is increased by conditioned media from the cultures of the macrophages grown either in the presence of DMOG or on PCLF/DMOG. Collectively, these results indicate that PCLF/DMOG meshes promote impaired wound healing in diabetic rats by modulating macrophage responses, enhancing angiogenesis and nerve innervation, and improving ECM synthesis.

We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor β (TGFβ)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.

The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

OBJECTIVE

We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity.

METHODS

HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR.

RESULTS

HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression.

CONCLUSIONS

HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. It contains an EGF-like domain as well as a heparin-binding domain that allows for interactions with heparin and cell-surface heparan sulfate. Soluble mature HB-EGF, a ligand of human epidermal growth factor receptors 1 and 4, is cleaved from the membrane-associated pro-HB-EGF by matrix metalloproteinase or a disintegrin and metalloproteinase in a process called ectodomain shedding. Signaling through human epidermal growth factor receptors 1 and 4 results in a variety of effects, including cellular proliferation, migration, adhesion, and differentiation. HB-EGF levels increase in response to different forms of injuries as well as stimuli, such as lysophosphatidic acid, retinoic acid, and 17β-estradiol. Because it is widely expressed in many organs, HB-EGF plays a critical role in tissue repair and regeneration throughout the body. It promotes cutaneous wound healing, hepatocyte proliferation after partial hepatectomy, intestinal anastomosis strength, alveolar regeneration after pneumonectomy, neurogenesis after ischemic injury, bladder wall thickening in response to urinary tract obstruction, and protection against ischemia/reperfusion injury to many cell types. Additionally, innovative strategies to deliver HB-EGF to sites of organ injury or to increase the endogenous levels of shed HB-EGF have been attempted with promising results. Harnessing the reparatory properties of HB-EGF in the clinical setting, therefore, may produce therapies that augment the treatment of various organ injuries.

HB-EGF is a member of the EGF family of ligands that is initially synthesized as a membrane-bound growth factor termed, proHB-EGF. The membrane bound proHB-EGF undergoes extensive proteolytic processing by several metalloproteinases capable of stimulating cellular proliferation. Soluble, mature HB-EGF binds to and activates EGF receptors. HB-EGF is a critical molecular component to a number of normal physiological processes including but not limited to tissue injury and wound healing, reproduction, angiogenesis and recently, adipogenesis. Misexpression of HB-EGF is linked to tumor formation and cancer including hepatocellular, pancreatic, gastric, breast, colon and melanoma, gliomas and glioblastomas. HB-EGF is a likely tool for therapeutic approaches to enhance treatment of injuries as well as a target for prevention of several cancers and obesity.

The first reports of the influences of oxidized LDL (oxLDL) on cell function pertained to negative effects on cell growth-growth arrest, injury, and toxicity. Since these studies, it has become apparent that sublethal levels of oxLDL cause some, but not all, cells to proliferate. This review highlights the growth-promoting effects of oxLDL rather than its inhibitory or injurious effects. Smooth muscle cells (SMCs) and monocyte-macrophages proliferate after exposure to oxLDL; endothelial cells do not. Scavenger receptors are involved in the proliferative effects on monocyte-macrophages, whereas the effects of oxLDL on SMCs appear to be receptor independent. Lysophosphatidylcholine (lysoPC), and structurally related lipids are among the growth-promoting constituents of oxLDL. OxLDL exerts at least a part of its effects by inducing expression or causing the release of growth factors. OxLDL (or lysoPC) can cause the release of basic fibroblast growth factor (bFGF) from SMCs; oxLDL (or lysoPC) can induce heparin binding EGF-like growth factor (HB-EGF) synthesis and release from macrophages. An imposing array of changes in cytokine and growth factor expression and/or release can be imposed by oxLDL on a wide variety of cell types. These effects and the studies probing the cell signaling events leading to them are described.

OBJECTIVE

Expression of uPA mRNA is massively up-regulated in the stroma of poorly differentiated ovarian tumors. We hypothesized that this expression was induced by paracrine signals from the epithelial tumor cells, and established an in vitro model of ovarian cancer microenvironment to study intercellular cross-talk.

METHODS

ES-2 clear cell carcinoma cells were grown in tissue culture inserts in a double-chamber system with fibroblastic stromal LEP cells embedded in Matrigel. Binding-site directed antibodies were used to neutralize soluble cytokines in ES-2 conditioned medium (CM) before incubation with LEP cells. Real time PCR measured uPA mRNA in LEP cells, as well as mRNA for cytokines in both cell types.

RESULTS

Co-culture with ES-2 cells as well as incubation with ES-2 CM induced uPA mRNA in LEP cells about two-fold. In short time (12 h) incubation of LEP cells with CM, antibodies to EGF and bFGF reduced induction of uPA mRNA, suggesting that these cytokines function as paracrine signals. EGF mRNA and bFGF mRNA were also found in ES-2 cells. At longer incubation (24 h) antibodies to bFGF, HB-EGF, HGF, IGF-1, and IL-1alpha reduced uPA mRNA induction, suggesting an autocrine function for these cytokines in LEP cells. In fact, expression of the same five cytokines was up-regulated in LEP cells exposed to CM.

CONCLUSIONS

We identified two cytokines as paracrine signals, and five cytokines as autocrine signals in ovarian cancer cell induced up-regulation of uPA mRNA in stromal fibroblastic cells. It is crucial to understand intra-tumoral cross-talk, since it can offer new therapeutic approaches.

Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

OBJECTIVE

Heparin-binding EGF-like growth factor (HB-EGF) protects the intestine from damage in animals. Future clinical trials of HB-EGF may involve administration of repeated doses of HB-EGF. Since HB-EGF activates EGF receptors which have been implicated in tumor development, we examined the effects of HB-EGF overexpression in the intestine.

METHODS

We generated transgenic (TG) mice in which the human HB-EGF gene is driven by the villin promoter to overexpress HB-EGF along the crypt-villous axis from the duodenum to the colon.

RESULTS

HB-EGF TG mice have increased enterocyte proliferation balanced by increased enterocyte apoptosis. Despite prolonged overexpression of HB-EGF, no evidence of intestinal hyperplasia or tumor formation occurs. Although HB-EGF TG mice have no significant phenotypic alterations under basal conditions, they have increased resistance to intestinal injury.

CONCLUSIONS

Prolonged intestinal HB-EGF overexpression results in no significant phenotypic alterations under basal conditions, but confers protection against intestinal injury.

OBJECTIVE

We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from multiple forms of injury via direct cytoprotective effects on the intestinal mucosa. In this study, we examined the effects of HB-EGF on the hemodynamics of intestinal arterioles, the major resistance vessels that regulate blood flow to the intestines, as an additional mechanism of HB-EGF-mediated intestinal protection.

METHODS

The hemodynamic effects of HB-EGF in rodent terminal mesenteric arterioles and human submucosal arterioles were examined ex vivo using a video dimension analyzer. Cultured human intestinal microvascular endothelial cells (HIMEC) were used to elucidate the mechanisms of HB-EGF-induced vasodilation.

RESULTS

HB-EGF significantly increased vessel diameter under conditions of increasing intraluminal pressure and increased flow rate. These HB-EGF-mediated vasodilatory effects were observed in terminal mesenteric arterioles from adult rats and 3 day old rat pups. These effects were confirmed in submucosal arterioles from human intestine. Furthermore, HB-EGF significantly reduced endothelin-1-induced mesenteric arteriolar vasoconstriction. The vasodilatory effects of HB-EGF were blocked by ET(B) receptor antagonism in adult rat arterioles, and also by nitric oxide synthase inhibition in rat pup and human infant arterioles. In HIMEC, HB-EGF significantly increased endothelin B (ET(B)) receptor protein expression and provoked intracellular calcium mobilization.

CONCLUSIONS

HB-EGF is a potent vasodilator of the intestinal microvasculature, further supporting its use in diseases manifested by decreased intestinal blood flow, including necrotizing enterocolitis.

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.

The mammalian kidney is susceptible to injury by ischemia/reperfusion and toxins, and regeneration after injury is characterized by hyperplasia and recovery of the damaged epithelial cells that line the tubules. Locally produced growth factors may serve as mediators of nephrogenesis and differentiation during renal development and of renal regeneration after acute injury. In cultured cells, administration of one or a mixture of growth factors to quiescent cells will initiate progression through the cell cycle and cell division. In the adult kidney, cell division normally is very low, but will increase up to 10-fold after acute injury. In addition to proliferation after lethal injury, there also is cellular repair in cells that have undergone sublethal injury. Recent studies indicate that growth factors inhibit programmed cell death in response to acute injury. Growth factors also may initiate or promote protein and lipid biosynthesis and provide an intracellular milieu that promotes cellular repair. In addition to cellular repair, growth factors also may be involved in the re-establishment of cell-extracellular matrix and cell-cell integrity. Finally, growth factors may limit injury by decreasing the factors that induce damage. Increased local renal expression of growth factors in response to acute injury include heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I), transforming growth factor-beta, parathyroid hormone-related peptide, and acidic fibroblast growth factor. In a number of experimental models of acute renal injury, administration of exogenous growth factors has been shown to accelerate both structural and functional recovery. Specifically, EGF, IGF-1, and HGF all have been shown to be effective in this regard. These studies are reviewed and potential therapeutic uses of growth factors and cytokines will be discussed.

Estrogen receptor-α (ER) targeted therapies are routinely used to treat breast cancer. However, patient responses are limited by resistance to endocrine therapy. Breast cancer cells resistant to the pure steroidal ER antagonist fulvestrant (fulv) demonstrate increased activation of epidermal growth factor receptor (EGFR) family members and downstream ERK signaling. In this study, we investigated the effects of fulv on EGFR signaling and ligand regulation in several breast cancer cell lines. EGFR/HER2/HER3 phosphorylation and ERK1,2 activation were seen after 24-48 h after fulvestrant treatment in ER-positive breast cancer cell lines. 4-Hydroxy-tamoxifen and estradiol did not cause EGFR activation. Fulvestrant did not affect EGFR expression. Cycloheximide abolished the ability of fulv to activate EGFR suggesting the autocrine production of EGFR ligands might be responsible for fulvestrant induced EGFR signaling. qRT-PCR results showed fulv differentially regulated EGFR ligands; HB-EGF mRNA was increased, while amphiregulin and epiregulin mRNAs were decreased. Fulvestrant induced EGFR activation and upregulation of EGFR ligands were ER dependent since fulv treatment in C4-12, an ER-negative cell line derivative of MCF-7 cells, did not result in EGFR activation or change in ligand mRNA levels. ER downregulation by siRNA induced similar EGFR activation and regulation of EGFR ligands as fulvestrant. Neutralizing HB-EGF antibody blocked fulv-induced EGFR activation. Combination of fulv and EGFR family tyrosine kinase inhibitors (erlotinib and lapatinib) significantly decreased EGFR signaling and cell survival. In conclusion, fulvestrant-activated EGFR family members accompanied by ER dependent upregulation of HB-EGF within 48 h. EGF receptor or ligand inhibition might enhance or prolong the therapeutic effects of targeting ER by fulvestrant in breast cancer.

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, "decoy" antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

The transmembrane glycoprotein CD44 is currently thought to be the main cell surface receptor for the glycosaminoglycan hyaluronate. We previously showed that (1) CD44 regulate keratinocyte proliferation; (2) topical retinoids dramatically increase the expression of CD44, hyaluronate and hyaluronate synthase (HAS)s in mouse epidermis; (3) topical retinaldehyde restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus lesions; and (4) retinaldehyde-induced proliferative response of keratinocytes is a CD44-dependent phenomenon and requires the presence of HB-EGF, erbB1 and matrix metalloproteinases. In this study, we analyzed the effect of UV irradiation on the levels of epidermal hyaluronate and CD44 in mice, as well as its potential prevention by topical retinoids. UVA (10 J/cm(2)) or UVB (1 J/cm(2)) irradiation significantly decreased the expression of CD44 and hyaluronate in the epidermis of hairless mice after 2 h. Expression of both epidermal CD44 and hyaluronate was reconstituted within 24 h. Topical application of retinaldehyde for 3 days prior to UVA or UVB irradiation prevented the decrease of CD44 and hyaluronate expression. Topical retinol and retinoic acid also increased the basal levels of epidermal CD44 and hyaluronate, although their preventive effect on UV-induced decrease of these molecules was less pronounced as compared to topical retinaldehyde. These data confirm the relationships between retinoid and CD44 pathways, although the primary target(s) of UV leading to CD44 and hyaluronate degradation remain to be elucidated.

Under diabetic conditions, the Maillard reaction facilitates the production of reactive oxygen species, and the activity of antioxidant enzymes such as Cu,Zn-superoxide dismutase is decreased, resulting in a remarkable increase of oxidative stress. The oxidative stress attacks DNA, lipids, and proteins and is also thought to be involved in the pathogenesis of diabetic complications, including the progression of macroangiopathy. Proliferation of smooth muscle cells (SMCs) is known to be associated with progression of macroangiopathy and is modulated by several growth factors. At least three mitogens for SMCs, platelet-derived growth factor (PDGF), fibroblast growth factor, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are known to be produced by SMCs themselves and are considered to be the most potent growth factors in the progression of macroangiopathy as seen in diabetes. HB-EGF, but not PDGF, is regulated at the transcriptional level by 3-deoxyglucosone (3-DG), a major and highly reactive intermediate in the glycation reaction. The induction seems to be triggered by the increase of reactive oxygen species produced by 3-DG. Taken together, glycation reactions under diabetic conditions may be highly associated with the pathogenesis of diabetic macroangiography by enhancing the gene expression of HB-EGF.

The induction of prostaglandin G/H synthase (PGHS; prostaglandin endoperoxide synthase, cyclooxygenase) by proinflammatory cytokines accounts, at least in part, for the altered eicosanoid biosynthesis in inflammatory diseases. In secondary cultures of normal human bronchial epithelial cells (NHBECs), interferon-gamma (IFN-gamma, 10 ng/ml for 24 h) increased the amount of prostaglandin E2 (PGE2) released in response to stimulation with exogenous arachidonic acid (5 microM). The enhanced production of PGE2 reflected the upregulation of PGHS-2 as indicated by enhanced expression of PGHS-2 RNA and increased recovery of PGHS-2 protein in NHBECs. IFN-gamma did not alter the production of PGE2 in A549 cells (a human lung adenocarcinoma cell line) or 6-keto-PGF1alpha in human umbilical vein endothelial cells (HUVECs), although prostaglandin release and/or the expression of PGHS-2 RNA in these cell lines was upregulated by other proinflammatory cytokines. Induction of PGHS-2 RNA in IFN-gamma-treated NHBECs, which peaked at 24 h, suggested the presence of an intermediary substance regulating the expression of PGHS-2. When the binding between the epidermal growth factor (EGF) receptor and its ligands was disrupted by a neutralizing antibody (LA-1), IFN-gamma failed to upregulate the release of PGE2 and the expression of PGHS-2 RNA in NHBECs. Furthermore, IFN-gamma induced the expression of RNAs for a number of ligands at the EGF receptor TGF-alpha; heparin-binding EGF-like growth factor (HB-EGF); and amphiregulin in NHBECs, and when administered exogenously, these ligands increased PGE2 release from NHBECs. Heparin at the concentration that neutralized the function of amphiregulin, or antibodies against TGFalpha or HB-EGF also reduced the release of PGE2 from IFN-gamma-stimulated NHBECs. These data are consistent with the presence of an autocrine growth factor/EGF receptor loop regulating PGHS-2 expression and PGE2 synthesis in bronchial epithelial cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) cDNA was isolated from a subtracted cDNA library that selected for progesterone-induced transcripts from rat uterine stromal cells. In this study, the effects of progesterone and estradiol on the expression of HB-EGF in mature rat uterine epithelial and stromal cells have been examined. RNase protection assays and in situ hybridization demonstrated that progesterone stimulated expression of HB-EGF in rat uterine stromal cells, but repressed levels of HB-EGF mRNA in luminal and glandular epithelial cells. In contrast, estradiol treatment strongly enhanced HB-EGF expression in epithelial cells, but had no effect on mRNA levels for this growth factor in stromal cells. Progesterone treatment followed by estradiol injection stimulated HB-EGF expression in stromal cells and repressed expression in luminal and glandular epithelium. Stimulation of HB-EGF expression in stromal cells by progesterone was not inhibited by treatment with cycloheximide, demonstrating that HB-EGF mRNA expression is a primary response of stromal cells to progesterone. These results reveal that expression of HB-EGF is stimulated in epithelial and stromal cells in vivo under the same hormonal conditions that induce cell proliferation in each of these cell types and strongly suggest that HB-EGF may mediate the mitogenic effects of steroid hormones in the rat uterus.

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.