Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling.

Patients diagnosed with melanoma have a poor prognosis due to regional invasion and metastases. The receptor tyrosine kinase epidermal growth factor receptor (EGFR) is found in a subtype of melanoma with a poor prognosis and contributes to drug resistance. Aloysia citrodora essential oil (ALOC-EO) possesses an antitumor effect. Understanding signaling pathways that contribute to the antitumor of ALOC-EO is important to identify novel tumor types that can be targeted by ALOC-EO. Here, we investigated the effects of ALOC-EO on melanoma growth and tumor cell migration. ALOC-EO blocked melanoma growth in vitro and impaired primary tumor cell growth in vivo. Mechanistically, ALOC-EO blocked heparin-binding-epidermal growth factor (HB-EGF)-induced EGFR signaling and suppressed ERK1/2 phosphorylation. Myelosuppressive drugs upregulated HB-EGF and EGFR expression in melanoma cells. Cotreatment of myelosuppressive drugs with ALOC-EO improved the antitumor activity and inhibited the expression of matrix metalloproteinase-7 and -9 and a disintegrin and metalloproteinase domain-containing protein9. In summary, our study demonstrates that ALOC-EO blocks EGFR and ERK1/2 signaling, with preclinical efficacy as a monotherapy or in combination with myelosuppressive drugs in melanoma.

Oxytocin Influences Male Sexual Activity via Non-synaptic Axonal Release in the Spinal Cord.

Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the "spinal ejaculation generator (SEG)." We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion-a localized volume transmission-to reach oxytocin receptors on GRP neurons and facilitate male sexual function.

Hyaluronan/collagen hydrogels containing sulfated hyaluronan improve wound healing by sustained release of heparin-binding EGF-like growth factor.

Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72 h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings.

Ablation of Foxl1-Cre-labeled hepatic progenitor cells and their descendants impairs recovery of mice from liver injury.

BACKGROUND & AIMS: Foxl1(+) hepatic progenitor cells (HPCs) differentiate into cholangiocytes and hepatocytes after liver injury. We investigated the requirement for Foxl1(+) HPCs in recovery from liver injury in mice. METHODS: We developed mice in which we could trace and delete Foxl1-expressing HPCs and their descendants (Foxl1-Cre;Rosa(YFP/iDTR)-inducible diphtheria toxin receptor [iDTR] mice). Foxl1-Cre-negative mice were used as controls. Liver damage was induced in male mice by placing them on choline-deficient, ethionine-supplemented (CDE) diets for 15 days; mice then were placed on normal diets and allowed to recover. Liver damage was induced in female mice by placing them on 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diets, followed by a recovery period. Some mice were given injections of diphtheria toxin during the recovery phase to delete Foxl1-Cre-marked HPCs and their descendants. Livers were collected from all mice and analyzed by immunofluorescence, quantitative reverse-transcription polymerase chain reaction, flow cytometry, and histologic analyses. RESULTS: Foxl1-Cre-marked HPCs were required for the development of cholangiocytes and hepatocytes in livers after CDE diet-induced injury. A smaller percentage of yellow fluorescent protein-positive (YFP(+)) hepatocytes contained markers of oxidative stress, DNA damage, or cell death than YFP-negative hepatocytes, indicating that YFP(+) hepatocytes are newly formed cells. Injection of diphtheria toxin deleted YFP(+) cells from Foxl1-Cre;Rosa(YFP/iDTR) mice and prevented the resolution of hepatic steatosis. In mice recovering from DDC diet-induced injury, most cholangiocytes arose from Foxl1-Cre-marked HPCs. Deletion of YFP(+) cells did not alter levels of markers of liver injury or liver function. CONCLUSIONS: Based on studies of Foxl1-Cre;Rosa(YFP/iDTR) mice, Foxl1(+) HPCs and/or their descendants are required for the development of cholangiocytes and hepatocytes in liver after CDE diet-induced injury.

Hyalurosome gene regulation and dose-dependent restoration of skin atrophy by retinaldehyde and defined-size hyaluronate fragments in dermatoporosis.

BACKGROUND: Dermatoporosis is an emerging clinical condition caused by chronological skin aging, long-term sun exposure and chronic use of corticosteroids; however, genomic expression in dermatoporosis and the efficacy of different therapeutic approaches to prevent and treat dermatoporosis have not been investigated so far. OBJECTIVE: We examined the possible effect of topical retinaldehyde (RAL) and defined-size hyaluronate fragments (HAFi) on the expression of hyalurosome genes potentially involved in the pathogenesis of dermatoporosis. We also explored the effect of different concentrations of HAFi on skin thickness. METHODS: 13 persons were separated into a young control group (n = 8) and a dermatoporosis group (n = 5). Topical treatment of both groups with a combination of 0.05% RAL and 1 or 0.2% HAFi was applied on the forearm twice daily for 30 days. Forearm skin biopsies of both groups were performed before and after application. Hyalurosome genes CD44, heparin-binding epidermal growth factor (HB-EGF), ErbB1, hyaluronate synthase 3 (HAS3) and Hyal2 were chosen as potential markers of dermatoporosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for quantification of mRNA expression of the target hyalurosome genes. Measurement of forearm skin thickness before and after treatment was performed by ultrasonography. Analysis of the results was done by Student's t test. A p value <0.05 was considered statistically significant. RESULTS: In qRT-PCR analysis the relative expression of hyalurosome (CD44, HAS3, HB-EGF) genes was found to be reduced in patients prior to topical treatment and to be notably increased following treatment. The reduced expression of CD44 and HAS3 in patients was specifically restored in dermatoporotic patients after treatment. No difference in skin thickness was observed in controls after treatment. The treatment caused a significant increase in skin thickness in dermatoporotic patients. This increase was more significant with 1% HAFi when compared to 0.2% HAFi. RAL and HAFi also caused a significant reduction in purpuric lesions in patients with dermatoporosis. CONCLUSION: Our results indicate that topically applied RAL and HAFi regulate hyalurosome gene expression in dermatoporosis and that they show a dose-dependent effect on the correction of skin atrophy in dermatoporotic patients.

Ablation of Sim1 neurons causes obesity through hyperphagia and reduced energy expenditure.

Single-minded 1 (Sim1) is a transcription factor necessary for development of the paraventricular nucleus of the hypothalamus (PVH). This nucleus is a critical regulator of appetite, energy expenditure and body weight. Previously we showed that Sim1(+/-) mice and conditional postnatal Sim1(-/-) mice exhibit hyperphagia, obesity, increased linear growth and susceptibility to diet-induced obesity, but no decrease in energy expenditure. Bilateral ablation of the PVH causes obesity due to hyperphagia and reduced energy expenditure. It remains unknown whether Sim1 neurons regulate energy expenditure. In this study, Sim1cre mice were bred to homozygous inducible diphtheria toxin receptor (iDTR) mice to generate mice expressing the simian DTR in Sim1 cells. In these mice, Sim1 neuron ablation was performed by intracerebroventricular (ICV) injection of diphtheria toxin. Compared to controls, mice with Sim1 neuron ablation became obese (with increased fat mass) on a chow diet due to increased food intake and reduced energy expenditure. In post-injection mice, we observed a strong inverse correlation between the degree of obesity and hypothalamic Sim1 expression. The reduction in baseline energy expenditure observed in these mice was accompanied by a reduction in activity. This reduction in activity did not fully account for the reduced energy expenditure as these mice exhibited decreased resting energy expenditure, decreased body temperature, decreased brown adipose tissue temperature, and decreased UCP1 expression suggesting an impairment of thermogenesis. In injected mice, hypothalamic gene expression of Sim1, oxytocin (OXT) and thyrotropin releasing hormone (TRH) was reduced by about 50%. These results demonstrate that Sim1 neurons in adult mice regulate both food intake and energy expenditure. Based on the body of work in the field, feeding regulation by Sim1 neurons likely occurs in both the PVH and medial amygdala, in contrast to energy expenditure regulation by Sim1 neurons, which likely is localized to the PVH.

Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.

Heparin-binding epidermal growth factor-like growth factor downregulates expression of activator protein-1 transcription factor after intestinal ischemia-reperfusion injury.

The pathogenesis of necrotizing enterocolitis (NEC) is unknown. Ischemia and reperfusion (I/R) injury have been considered to be major contributing factors. More recent reports have noted that apoptosis is a significant and perhaps the principal contributor to cell death after I/R injury. Recent studies have revealed that activator protein 1 (AP-1) family proteins including c-Fos and c-Jun potentially induce either the proliferation or apoptosis of the cells in the brain, heart, kidney, and liver. c-Fos and c-Jun expression has also been reported to be upregulated in postischemic intestinal epithelial cells (IECs). Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a potent cytoprotective factor in various pathologic conditions and plays a pivotal role in mediating the earliest cellular responses to injury. This study aims to examine whether HB-EGF, a proven intestinal cytoprotective molecule, exerts its protective effects through modulation of AP-1 transcription factor after intestinal I/R injury. Thirty rats were randomly divided into the following 5 groups: (1) normal control group; (2) ischemia group; (3) I/R group; (4) ischemia group with HB-EGF (400 µg/kg), and (5) I/R group with HG-EGF (400 µg/kg). c-Fos and c-Jun messenger RNAs and protein levels were determined by real-time quantitative polymerase chain reaction (PCR) and Western analyses, respectively. Statistical analysis was performed using ANOVA with Dunn's test. The messenger RNA levels of the c-Fos and c-Jun increased after intestinal ischemia or the intestinal reperfusion phase. HB-EGF pretreatment significantly decreased c-Fos and c-Jun messenger RNAs. The expression of protein levels of c-Fos and c-Jun were correlation with the expression of messenger RNA level. HB-EGF intestinal cytoprotection is mediated, in part, by downregulation of the expression of AP-1 transcription factor after intestinal I/R injury.

Heparin-binding epidermal growth factor expression in KATO-III cells after Helicobacter pylori stimulation under the influence of strychnos Nux vomica and Calendula officinalis.

INTRODUCTION: Previous studies have shown the stimulating effect of Helicobacter pylori on the gene expression of heparin-binding epidermal growth factor (HB-EGF) using the gastric epithelial cell line KATO-III. Strychnos Nux vomica (Nux vomica) and Calendula officinalis are used in highly diluted form in homeopathic medicine to treat patients suffering from gastritis and gastric ulcers. AIM AND METHOD: To investigate the influence of Nux vomica and Calendula officinalis on HB-EGF-like growth factor gene expression in KATO-III cells under the stimulation of H. pylori strain N6 using real-time PCR with and without addition of Nux vomica and Calendula officinalis as a 10c or 12c potency. RESULTS: Baseline expression and stimulation were similar to previous experiments, addition of Nux vomica 10c and Calendula officinalis 10c in a 43% ethanolic solution led to a significant reduction of H. pylori induced increase in gene expression of HB-EGF (reduced to 53.12+/-0.95% and 75.32+/-1.16% vs. control; p<0.05), respectively. Nux vomica 12c reduced HB-EGF gene expression even in dilutions beyond Avogadro's number (55.77+/-1.09%; p<0.05). Nux vomica 12c in a 21.5% ethanol showed a smaller effect (71.80+/-3.91%, p<0.05). This effect was only be observed when the drugs were primarily prepared in ethanol, not in aqueous solutions. The data suggest that both drugs prepared in ethanolic solution are potent inhibitors of H. pylori induced gene expression.

Suppression of proHB-EGF carboxy-terminal fragment nuclear translocation: a new molecular target therapy for gastric cancer.

PURPOSE: Inactivation of epidermal growth factor (EGF) receptor (EGFR) represents a promising strategy for the development of selective therapies against epithelial cancers and has been extensively studied as a molecular target for cancer therapy. However, little attention has been paid to remnant cell-associated domains created by cleavage of EGFR ligands. The present study focused on recent findings that cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), an EGFR ligand, induces translocation of the carboxyl-terminal fragment (CTF) of HB-EGF from the plasma membrane to the nucleus and regulates cell cycle. EXPERIMENTAL DESIGN: Two gastric cancer cell lines, MKN28 and NUGC4, were used. KB-R7785, an inhibitor of proHB-EGF shedding, was used to suppress HB-EGF-CTF nuclear translocation with cetuximab, which inhibits EGFR phosphorylation. Cell growth was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, apoptosis was evaluated by assay of caspase-3 and caspase-7, and cell cycle was investigated by flow cytometry. RESULTS: Immunofluorescence study confirmed that KB-R7785 inhibited HB-EGF-CTF nuclear translocation under conditions of proHB-EGF shedding induction by 12-O-tetradecanoylphorbol-13-acetate in gastric cancer cells. KB-R7785 inhibited cell growth in a dose-dependent manner and high-dose KB-R7785 induced apoptosis. Moreover, KB-R7785 induced cell cycle arrest and increased sub-G1 DNA content. KB-R7785 suppressed cyclin A and c-Myc expression. All effects of KB-R7785 were reinforced by combination with cetuximab. CONCLUSIONS: These results suggest that both inhibition of EGFR phosphorylation and inhibition of HB-EGF-CTF nuclear translocation play crucial roles in inhibitory regulation of cancer cell growth. Suppression of HB-EGF-CTF nuclear translocation might offer a new strategy for treating gastric cancer.

Apple polyphenol extracts prevent aspirin-induced damage to the rat gastric mucosa.

Aspirin causes gastroduodenal ulcers and complications. Food bioactive compounds could exert beneficial effects in the gastrointestinal tract. We evaluated whether apple polyphenol extract (APE) reduced aspirin-induced injury to the rat gastric mucosa. Rats were treated with APE (10(-4) m catechin equivalent) before oral aspirin (200 mg/kg). Cyclo-oxygenase-2 (COX-2), transforming growth factor-alpha (TGF alpha) and heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA and protein expression were assessed by RT-PCR and Western blot analysis, respectively; malondialdehyde (MDA) was determined by HPLC; gastric secretion was evaluated in pylorus-ligated rats. APE decreased acute and chronic aspirin injury both macroscopically and microscopically (approximately 50 % decrease in lesion score; P < 0.05). Aspirin up-regulated mRNA and protein expression of COX-2 and HB-EGF, but not of TGF alpha; APE reduced aspirin-induced mRNA and protein over-expression of COX-2 and HB-EGF; aspirin significantly increased gastric MDA and this effect was counteracted by APE pre-treatment. APE did not significantly affect gastric acid secretion. In conclusion, APE reduces aspirin-induced gastric injury independently of acid inhibition. We speculate that APE might be of therapeutic use in the prophylaxis of aspirin-related gastropathy.

[Effect of total flavones from Cuscuta chinensis on expression of Fas/FasL, PCNA and HB-EGF in SD rats model with bromocriptine-induced abortion].

OBJECTIVE: To explore the effect of total flavones from cuscuta chinensis (TFCC) on expression of Fas, PCNA and HB-EGF in SD rats model with bromocriptine-induced abortion. METHODS: The model rats of bromocriptine during 6-8 d of pregnancy induced early abortion was established, adopting respectively herbs in high and low dosage and progesterone affect model rat and after 12 d, Immunohistochemical was applied to determine Fas, HB-EGF and PCNA in deciduas and placenta. RESULTS: Expression of PCNA on trophoblast and deciduas, HB-EGF on trophoblast, PR on deciduas in the model used Semen cuscutae flavonoid, proesterone and normal pregnacy, were significantlly higher than those of the pure model. Expression of Fas on trophoblast and deciduas in above four groups, were significantlly lower than those of the pure model. There were no expression of HB-EGF on deciduas. CONCLUSION: TFCC regulates the proliferation and apoptosis of the deciduas and cytotrophoblasts and prevents spontaneous abortions.

Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo. Berberine improves neointima formation in a rat model.

Berberine, an alkaloid isolated from Chinese medicinal herbs, long been known for its anti-microbial activity and used to treat various infectious disorders in traditional Chinese medicine. In the present study, we have tested the hypothesis that berberine could inhibit vascular smooth muscle cell (VSMC) proliferation as it did in endothelial cells or cancer cells. Our results show that berberine significantly inhibits growth factor, mainly angiotensin II (AngII) and heparin binding epidermal growth factor (HB-EGF), induced VSMC proliferation and migration in vitro, and this effect is achieved by delaying or partially suppressing activation of Akt pathway rather than ERK pathway. Furthermore, we have examined its effect in vivo using a rat carotid artery injury model. A 28 days of chronic berberine treatment using an osmotic pump (100 microg kg(-1)d(-1), 2 weeks before and 2 weeks after the injury) improved neointima formation. The Neointima/Media ratio for control group and berberine treated group were 1.14+/-0.11 and 0.85+/-0.06 (p<0.05), respectively, and the reduction was approximately 25%. The result of the present study suggests a possibility of berberine being a potent agent to control restenosis after balloon angioplasty and warrants further study to gain a more complete understanding of its underlying mechanisms at a cellular level.

Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells.

BACKGROUND: The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells. METHODS: Isolated cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. RESULTS: Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types. CONCLUSIONS: The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.

Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation.

Delayed implantation (embryonic diapause) occurs when the embryo at the blastocyst stage achieves a state of suspended animation. During this period, blastocyst growth is very slow, with minimal or no cell division. Nearly 100 mammals in seven different orders undergo delayed implantation, but the underlying molecular mechanisms that direct this process remain largely unknown. In mice, ovariectomy before preimplantation ovarian estrogen secretion on day 4 of pregnancy initiates blastocyst dormancy, which normally lasts for 1-2 weeks by continued progesterone treatment, although blastocyst survival decreases with time. An estrogen injection rapidly activates blastocysts and initiates their implantation in the progesterone-primed uterus. Using this model, here we show that among approximately 20,000 genes examined, only 229 are differentially expressed between dormant and activated blastocysts. The major functional categories of altered genes include the cell cycle, cell signaling, and energy metabolic pathways, particularly highlighting the importance of heparin-binding epidermal growth factor-like signaling in blastocyst-uterine crosstalk in implantation. The results provide evidence that the two different physiological states of the blastocyst, dormancy and activation, are molecularly distinguishable in a global perspective and underscore the importance of specific molecular pathways in these processes. This study has identified candidate genes that provide a scope for in-depth analysis of their functions and an opportunity for examining their relevance to blastocyst dormancy and activation in numerous other species for which microarray analysis is not available or possible due to very limited availability of blastocysts.

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor.

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.

Effect of heparin on tissue binding activity of fibroblast growth factor and heparin-binding epidermal growth factor in experimental colitis in rats.

There have been several reports implying a benefit for heparin therapy in patients with refractory ulcerative colitis. Although this effect has been attributed to the anti-inflammatory properties of heparin, other mechanisms have not been excluded. Heparin is a potent modulator of receptor binding of growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin-binding epidermal growth factor (HB-EGF), that play a role in wound repair. We examined the effect of heparin on the functional levels of FGF and HB-EGF in a model of experimental colitis. Fifty-six Wistar rats were divided into four groups: group 1 was the control group, group 2 received s.c. heparin 50 units/kg/d, group 3 underwent induction of 3% iodoacetamide colitis, and group 4 underwent induction of colitis and heparin treatment. Rats were killed and evaluated for severity of colitis by macroscopic and microscopic colitis scores, area of inflammation, and myeloperoxidase levels. FGF and HB-EGF levels were functionally assessed in colonic tissue in each group. Heparin therapy resulted in significant improvement in macroscopic and microscopic features of colitis (p < 0.05), accompanied by a partial reduction in myeloperoxidase levels. FGF receptor binding activity was identical in groups 1 and 2 but increased more than 3-fold after colitis induction in group 3 (p < 0.05). Treatment with heparin caused a significant decrease in FGF concentration. Levels of HB-EGF binding activity were similar in groups 1 and 2 and decreased in group 3 (p < 0.01). Heparin caused a significant increase in HB-EGF content in group 4 (p < 0.05). Levels of growth factors are altered differently in experimental colitis. Colonic FGF binding activity increases with colitis, whereas HB-EGF binding decreases with colitis. These trends were reversed by heparin, concomitant with a clinical and pathologic improvement in colitis. We suggest that one mechanism of heparin-mediated improvement in colitis may involve tissue healing associated with changes in functional levels of colonic growth factors.

Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells.

BACKGROUND & AIMS: Epidermal growth factor (EGF)-like growth factors are induced after acute gastric injury and may play an important role in mucosal repair. However, the mechanisms that trigger these growth factors are poorly understood. We determined the role of EGF receptor (EGFR) in stress-induced expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric epithelial cell line (RGM1 cells). METHODS: RGM1 cells were transfected with a plasmid containing complementary DNA encoding a dominant-negative human EGFR (HERCD533). Cells were treated with hydrogen peroxide (0-400 micromol/L) or sorbitol (600 mmol/L). Tyrosine phosphorylation of EGFR was determined by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. HB-EGF messenger RNA and protein were determined with Northern and Western blotting, respectively. Cell growth was evaluated by cell number and [(3)H]thymidine incorporation. RESULTS: Oxidative stress and osmotic stress induced tyrosine phosphorylation of EGFR within 2 minutes, followed by a marked increase in HB-EGF and amphiregulin transcripts in RGM1 cells. Introduction of HERCD533 into the cells inhibited not only tyrosine phosphorylation of EGFR but also growth response to EGF. Furthermore, oxidative stress-induced HB-EGF messenger RNA expression was impaired in HERCD533-expressing cells. CONCLUSIONS: EGFR plays a crucial role in the stress-induced expression of EGF-like growth factors in gastrointestinal epithelial cells.

Percutaneous sacral third nerve root neurostimulation improves symptoms and normalizes urinary HB-EGF levels and antiproliferative activity in patients with interstitial cystitis.

OBJECTIVES: A highly effective treatment for interstitial cystitis (IC) remains elusive. We determined whether sacral third nerve root (S3) percutaneous neurostimulation (PNS) might be effective in relieving symptoms of IC, as well as in normalizing urinary factors that are specifically altered in IC. METHODS: Six consecutive patients with symptoms and cystoscopic findings compatible with IC underwent 5 days of continuous S3 neurostimulation by way of leads placed percutaneously into the S3 foramen. Patients filled out voiding frequency diaries and pain and urgency questionnaires before PNS and at the end of PNS when the leads were removed. Urine specimens were collected at these two time points and measured for heparin-binding epidermal growth factor-like growth factor (HB-EGF) by enzyme-linked immunosorbent assay and for antiproliferative factor (APF) activity by (3)H-thymidine uptake by normal human bladder urothelial cells. RESULTS: S3 PNS significantly improved all measured parameters toward normal values. Voiding frequency decreased twofold from 23.1 +/- 4.6 to 10.6 +/- 4.0 voids daily during PNS (P = 0.0001). Pelvic pain on a scale of 1 to 10 decreased from 7.0 +/- 1.6 to 2.3 +/- 3.2 (P = 0.05). Urinary urgency on a scale of 1 to 10 decreased from 6.0 +/- 2.2 to 1.8 +/- 1.7 (P = 0. 02). Urinary HB-EGF concentration increased sevenfold from 1.5 +/- 2. 1 to 11.0 +/- 1.7 ng/mL (P <0.0001), and urinary APF activity decreased from -76.1% +/- 31% to -4.5% +/- 8.8% (P = 0.005). CONCLUSIONS: S3 PNS significantly decreased symptoms and normalized urinary HB-EGF and APF activity in patients with IC. These results suggest that permanent S3 PNS may be beneficial in treating IC.

Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study.

Dietary omega-3 fatty acids retard coronary atherosclerosis. Previously, we demonstrated that dietary omega-3 fatty acids reduce platelet-derived growth factor (PDGF)-A and PDGF-B mRNA levels in unstimulated, human mononuclear cells (MNCs). In a randomized, investigator-blinded intervention trial, we have now compared the effect of ingestion of 7 g/d omega-3, omega-6, or omega-9 fatty acids for 4 weeks versus no dietary intervention on PDGF-A, PDGF-B, heparin-bound epidermal growth factor (HB-EGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 gene expression in unstimulated MNCs and in monocytes that were adherence-activated ex vivo in a total of 28 volunteers. In unstimulated MNCs, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-10%, 31+/-13%, and 40+/-14%, respectively, after omega-3 fatty acid ingestion, as assessed by quantitative polymerase chain reaction (all P<0.05). In monocytes that were adherence-activated ex vivo for 4 and 20 hours, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-13%, 20+/-15%, and 30+/-8%, respectively (all P<0.05). Interleukin-10 and HB-EGF mRNA steady-state levels were not influenced by omega-3 fatty acid ingestion. Expression of all respective mRNAs in control volunteers or in those ingesting omega-6 or omega-9 fatty acids were not altered. We conclude that human gene expression for PDGF-A, PDGF-B, and MCP-1, factors thought relevant to atherosclerosis, is constitutive, is constant, and can be reduced only by dietary omega-3 fatty acids in unstimulated and adherence-activated monocytes.