Heparin-binding Epidermal Growth Factor Levels Research Articles

B7-H3 enhances colorectal cancer progression by regulating HB-EGF via HIF-1α.

B7-H3 (or CD276) represents an important costimulatory molecule expressed in many malignant solid tumors, including colorectal cancer (CRC). The receptor of B7-H3 is not known, and the intracellular function of B7-H3 remains obscure. Herein, we report that B7-H3 upregulated the epidermal growth factor heparin-binding epidermal growth factor (HB-EGF), likely by regulating hypoxia-inducible factor 1α (HIF-1α) and thereby promoting the progression of CRC. Lentiviral transfection was performed on CRC cells to establish stable low-B7-H3 expression cells. A mechanistic analysis with an Agilent human gene expression profiling chip was conducted on them. Clinical data and specimens were collected to detect the connection between B7-H3 and HB-EGF in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the messenger RNA (mRNA) level of B7-H3, HB-EGF, and HIF-1α. Chromatin immunoprecipitation (ChIP) quantitative real-time PCR was conducted. The protein level of HIF-1α and the phosphatidylinositide 3-kinases (PI3K)-protein kinase B (AKT) pathway were detected by western blot. HIF-1α was recovered by lentiviral transfection, and the HB-EGF mRNA levels, proliferation, invasion, and angiogenesis ability were detected. B7-H3 promoted tumor progression through HB-EGF and the PI3K-AKT pathway. As B7-H3 was downregulated, HB-EGF levels were significantly reduced simultaneously, a growth trend that was shown by both CRC cell lines and cancer tissues. In addition, B7-H3 and HB-EGF had significant associations with tumor-node-metastasis (TNM) stage and lymph node metastasis in 50 CRC patients. The binding ability of HIF-1α to the HB-EGF promoter region was significantly decreased in the shB7-H3 RKO group. Western blot revealed that PI3K, AKT, and mammalian target of rapamycin (mTOR) protein amounts and p-AKT and p-mTOR phosphorylation were also downregulated in shB7-H3 RKO cells, suggesting that B7-H3 may regulate HIF-1α via PI3K-AKT signaling. After recovery of the HIF-1α level by lentiviral transfection, the HB-EGF mRNA levels, proliferation, invasion, and angiogenesis in CRC cells recovered as well. B7-H3 may transmit intracellular signals through PI3K-AKT-mTOR-HIF-1α signaling, upregulating HB-EGF. As the final transcription factor of the pathway, HIF-1α regulates the transcription of the HB-EGF gene, thereby promoting HB-EGF expression, which eventually mediates cell proliferation, invasion, and angiogenesis and promotes the progression of CRC.

Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway

BACKGROUNDColorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway.AIMTo illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway.METHODSWe investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF.RESULTSGEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes AKT, RAF, KRAS expression levels. After knockdown of FOXQ1 in CRC cell lines, cell proliferation, migration and invasion were attenuated. Adding HB-EGF restored the migration and invasion ability of CRC, but not the cell proliferation ability. Kaplan–Meier survival analysis results showed that the combination of FOXQ1 and HB-EGF may serve to predict CRC survival.CONCLUSIONBased on these collective data, we propose that FOXQ1 promotes the invasion and metastasis of CRC via the HB-EGF/EGFR pathway.

Serum Amphiregulin and Heparin-Binding Epidermal Growth Factor as Biomarkers in Patients with Idiopathic Inflammatory Myopathy.

Background. The epidermal growth factors amphiregulin (AREG) and heparin-binding epidermal growth factor (HB-EGF) are implicated in the pathogenesis of several autoimmune diseases, but their clinical and pathological roles in idiopathic inflammatory myopathy (IIM) are unclear. Methods. Serum AREG and HB-EGF levels were measured by ELISA in patients with IIM (n = 37), systemic sclerosis (n = 17), and rheumatoid arthritis (n = 10), and for seven age- and sex-matched healthy controls (HCs). Associations between serum AREG or HB-EGF levels and the clinical parameters were analyzed. Results. Serum AREG levels in IIM patients were significantly elevated compared to those in HCs (median, 20.7 and 10.7 pg/mL, respectively; p = 0.025). In particular, serum AREG levels in IIM patients with interstitial lung disease (ILD) were higher than those of HCs (22.4 pg/mL, p = 0.027). The disease duration in patients with elevated serum AREG levels was significantly shorter compared to those who had normal serum AREG levels (7 and 21 months, respectively; p = 0.0012). Serum HB-EGF levels were significantly increased in IIM patients with elevated CK levels (136.2 pg/mL; p = 0.020) and patients with anti-Mi-2 antibody (183.7 pg/mL; p = 0.045) compared to those in HCs (74.9 pg/mL). Conclusion. These results suggested that AREG could be a promising biomarker associated with early-phase IIM-related ILD, and that HB-EGF expression was associated with muscle injury and regeneration in IIM.

Relationship of potential biomarkers of interstitial cystitis/painful bladder syndrome

BACKGROUND: Biomarker identification fulfills the need for simple, accurate, and non-invasive diagnostic methods for interstitial cystitis/painful bladder syndrome (IC/PBS). The simultaneous study of cytokines and growth factors is essential for understanding the role of biomarkers.
 AIM: This work determines the concentration of interleukins (IL) 6 and 8, nerve growth factor (NGF), heparin-binding epidermal growth factor (HB-EGF), and their relationship in patients with IC/PBS.
 MATERIALS AND METHODS: This study examined 126 female patients with IC/PBS, with a mean age of 46.714.0 years. The disease duration averaged 6.02.8 years. The control group consisted of 20 women without IC/PBS, with a mean age of 35.39.7 years. The level of IL-6 and IL-8 in blood serum and urine was determined by ELISA. The NGF and HB-EGF levels were determined by ELISA using the appropriate test kits. Statistical analysis was performed using SPSS software version 15.0. The Students t-test and Spearmans correlation coefficient were calculated.
 RESULTS: In patients with IC/PBS, blood levels of IL-6 and IL-8 exceeded the control indicator by 65.99% (t=4.62, p=0.000) and by 67.14% (t=2.81, p=0.006), respectively, the NGF level exceeded it by 8.82% (t=0.10, p=0.920), and the HB-EGF level decreased by 30.84% (t=0.62, p=0.537). In urine, the level of IL-6 in IC/PBS was increased by 52.94% (t=7.62, p=0.000), IL-8 level was increased by 57.53% (t=2.01, p=0.049), NGF was increased by 50.44% (t=2.06, p=0.043), and HB-EGF level was increased by 56.44% (t=2.01, p=0.049). Cytokines correlated with growth factors with weakly bound in blood and urine (p 0.05). A strong, direct relationship was registered between the urine levels of NGF and HB-EGF (r=+0.987, p 0.05).
 CONCLUSIONS: Correlations between IL-6 and IL-8 with NGF and HB-EGF in urine indicate an increased risk of damage to the bladder epithelium. The increase in urinary HB-EGF is a response to a significant increase in urinary NGF and may be a bladder epithelial defense mechanism.

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway.

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.

Correlation of IL-1 and HB-EGF with endometrial receptivity.

Fluctuations of interleukin-1β (IL-1β) and heparin-binding epidermal growth factor (HB-EGF) in endometrial receptivity were detected. Seventy-two patients receiving in vitro fertilization-embryo transfer (IVF-ET) for the first time in Yantaishan Hospital from July 2015 to September 2015 due to infertility were selected. The serum and follicular fluid of patients in ovulation-promoting cycle were collected; the levels of IL-1β and HB-EGF in serum and follicular fluid were detected via enzyme-linked immunosorbent assay (ELISA), and the levels of serum estradiol (E2), progesterone (P), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected. The endometria in early follicular phase and middle luteal phase were collected, and the mRNA expression levels of IL-1β and HB-EGF were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Patients were divided into two groups, and the implantation group (n=33) and the non-implantation group (n=39), according to whether embryos were implanted and the general data. In IVF-ET, the levels of IL-1β and HB-EGF in follicular fluid and middle luteal phase, the level of serum IL-1β on human chorionic gonadotropin (HCG) day and embryo transfer (ET) day, the levels of E2, FSH and LH on HCG day in implantation group were obviously higher than those in non-implantation group (p<0.05); the level of P on ET day in implantation group was significantly higher than that in non-implantation group (p<0.05); the expression levels of IL-1β and HB-EGF in endometrium in middle luteal phase in implantation group were higher than those in non-implantation group (p<0.05); the expression levels of IL-1β and HB-EGF in endometrium were positively correlated with the levels of E2 and P, and endometrial thickness (p<0.05). IL-1β and HB-EGF may improve the endometrial receptivity to embryo, thus affecting the embryo implantation rate, through the synergistic action with E2 and P, so they may be the indexes of predicting the IVF-ET pregnancy outcome.

Blood-based markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

Circulating protein markers were assessed in patients with colorectal cancer (CRC) treated with cetuximab in CALGB 80203 to identify prognostic and predictive biomarkers. Patients with locally advanced or metastatic CRC received FOLFOX or FOLFIRI chemotherapy (chemo) or chemo in combination with cetuximab. Baseline plasma samples from 152 patients were analyzed for six candidate markers [epidermal growth factor (EGF), heparin‐binding EGF (HBEGF), epidermal growth factor receptor (EGFR), HER2, HER3, and CD73]. Analyte levels were associated with survival endpoints using univariate Cox proportional hazards models. Predictive markers were identified using a treatment‐by‐marker interaction term in the Cox model. Plasma levels of EGF, HBEGF, HER3, and CD73 were prognostic for overall survival (OS) across all patients (KRAS mutant and wild‐type). High levels of EGF predicted for lack of OS benefit from cetuximab in KRAS wild‐type (WT) patients (chemo HR = 0.98, 95% CI = 0.74–1.29; chemo+cetuximab HR = 1.54, 95% CI = 1.05–2.25; interaction P = 0.045) and benefit from cetuximab in KRAS mutant patients (chemo HR = 1.72, 95% CI = 1.02–2.92; chemo+cetuximab HR = 0.90, 95% CI = 0.67–1.21; interaction P = 0.026). Across all patients, higher HER3 levels were associated with significant OS benefit from cetuximab treatment (chemo HR = 4.82, 95% CI = 1.68–13.84; chemo+cetuximab HR = 0.95, 95% CI = 0.31–2.95; interaction P = 0.046). CD73 was also identified as predictive of OS benefit in KRAS WT patients (chemo HR = 1.28, 95% CI = 0.88–1.84; chemo+cetuximab HR = 0.60, 95% CI = 0.32–1.13; interaction P = 0.049). Although these results are preliminary, and confirmatory studies are necessary before clinical application, the data suggest that HER3 and CD73 may play important roles in the biological response to cetuximab.

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model

Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.

The Effect of Helicobacter pylori on Epidermal Growth Factor Receptor-Induced Signal Transduction and the Preventive Effect of Celecoxib in Gastric Cancer Cells

Background/AimsHelicobacter pylori infection induces cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) overexpression, and these factors may engage in cross-talk. The aim of the present study was to evaluate the effect of H. pylori on EGFR signaling pathways and to determine whether celecoxib has an inhibitory effect on this pathway.MethodsThe AGS cell line was cocultured with H. pylori G27 and the isogenic cagE- mutant. The expression of COX-2, EGFR, heparin binding-epidermal growth factor (HB-EGF), and transforming growth factor-β (TGF-β) was measured by real time-polymerase chain reaction (RT-PCR). Next, Western blot analyses of COX-2, EGFR, total Akt, phosphorylated Akt (pAkt), and phosphorylated glycogen synthase kinase-3β (pGSK3β) were performed after incubating H. pylori-treated AGS cells for 24 hours with various concentrations of celecoxib (0, 10, 20, and 30 µmol/L).ResultsH. pylori infection upregulated the mRNA levels of COX-2, EGFR, HB-EGF, and TGF-β, as detected by RT-PCR. However, AGS cells treated with cagE- mutants, which have a defective type IV secretion system, did not exhibit EGFR upregulation. Celecoxib had inhibitory effects on the H. pylori-induced overexpression of COX-2 (p=0.015), EGFR (p=0.025), pAkt (p=0.025), and pGSK3β (p=0.029) by Western blot analysis.ConclusionsH. pylori with an intact type IV secretion system activated the COX-2 and EGFR-Akt pathways in the AGS cell line. As celecoxib exhibited inhibitory effects on the EGFR signaling pathway, the cross-talk of COX-2 and EGFR likely mediates H. pylori-induced gastric cancer.

LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaPE (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Abstract 2129: HB-EGF expression in gastric cancer promotes chemotherapy resistance and represents a promising target for molecular therapy and a novel biomarker of 5-FU resistance

Abstract The incidence of gastric cancer (GC) has been on the decline in the U.S. over the last 40 years, but remains high in Asia and Latin America. The primary treatment for early-stage GC is surgery. Many patients will also benefit from adjuvant 5-fluorouracil (5-FU) or platin-based chemotherapy. When it presents at an advanced stage, GC uniformly carries a poor prognosis, due to chemotherapy resistance. To improve the response of GC to chemotherapy, molecular-targeted therapies are needed. One of the EGFR family ligands called heparin-binding epidermal growth factor (HB-EGF) was recently found to be up-regulated in several epithelial-based malignancies, including GC. Our objective in this study was to evaluate the role of HB-EGF in GC chemotherapy resistance. We hypothesize that constitutive expression of HB-EGF is associated with chemotherapy resistance. Furthermore we propose that inhibition of HB-EGF enhances drug sensitivity and augments the apoptotic response to chemotherapy in GC. To test our hypothesis, we isolated RNA and protein from a panel of 6 GC cell lines and measured the basal HB-EGF expression compared to that of normal gastric epithelia. We similarly measured HB-EGF expression in response to 5-FU or cisplatin treatment, and then determined the response when cells were co-treated with HB-EGF or an inhibitor of HB-EGF (CRM197). We found that HB-EGF expression at both the protein and RNA levels was highly variable across the panel of GC cell lines. In addition, HB-EGF expression increased in response to drug treatment at 24 and 48 hours post-treatment. A positive correlation was observed between HB-EGF mRNA levels and chemoresistance to 5-FU. GC cell lines that are high-expressers of HB-EGF showed decreased cell growth when treated with CRM197, whereas low-expressers increased when treated with recombinant HB-EGF. The combination of CRM197 with anticancer agents increased apoptosis in high-expressers, but made no significant difference in low-expressers. Likewise, HB-EGF gene silencing using a lentiviral short-hairpin RNA significantly decreased high-expressers survival and proliferation. Similar to our findings using GC cell lines, we found that human gastric cancer tumor samples showed highly variable HB-EGF expression compared to their matching normal gastric epithelia. Our findings suggest that HB-EGF plays an important role in the proliferation and chemotherapy resistance of a subset of gastric cancers. HB-EGF may represent a promising molecular target in those select patients, in whom the gastric cancer expresses high levels of HB-EGF. Furthermore, HB-EGF expression may represent a novel biomarker for 5-FU chemoresistance in patients with gastric cancer. 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 2129. doi:10.1158/1538-7445.AM2011-2129

Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

Background and aimsHelicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of...

Vegf And Hb-egf Expression And Localization In Chronically Overloaded Rat Plantaris And Soleus Muscle

Skeletal muscle hypertrophy is mediated by local mitogens that promote fiber growth and structural reorganization during this anabolic state. Two biochemicals that may participate in this process include Heparin-Binding Epidermal Growth Factor (HB-EGF) and Vascular Endothelial Growth Factor (VEGF). PURPOSE: To determine the expression profile and localization of HB-EGF and VEGF following functional overload (FO) of rat hindlimb muscles. METHODS: Bilateral FO for 3 and 7d (n= 5/group) of the plantaris and soleus muscles in adult female Sprague-Dawley rats was induced through surgical ablation of the gastrocnemius muscle and compared to normal controls (Con). Temporal protein expression levels were assessed through immunoblot analysis and localization was determined using immunofluorescent histochemistry. RESULTS: Absolute and relative (mg/g) soleus and plantaris muscle mass was increased (p<0.05) at 3 and 7d FO compared to Con. HB-EGF levels during FO were similar to Con. A muscle-specific response for VEGF expression occurred in the soleus and plantaris muscles at 3d FO: soleus VEGF decreased (p<0.05); conversely, a trend (p = 0.12) for elevated VEGF occurred in the plantaris. VEGF at 7d FO were similar to Con in both muscles. Immnunohistochemistry revealed a strong fiber membrane-specific localization for VEGF in both FO muscles. VEGF also appeared to be localized to fiber ends. CONCLUSION: VEGF expression profiles during FO were muscle-specific. The transient decrease of VEGF in the soleus during early FO may indicate a saturated VEGF state in this highly oxidative and vascular muscle. VEGF localization in proximity to the membrane suggests a role for vascular remodeling of the capillary network.

Differential distributions of peptides in the epidermal growth factor family and phosphorylation of ErbB1 receptor in adult rat brain

Using two-site enzyme immunoassays, we measured protein levels of epidermal growth factor (EGF), transforming growth factor alpha (TGFα), and heparin-binding epidermal growth factor (HB-EGF) in adult rat brain, and compared them with the phosphorylation levels of their receptor (ErbB1). There were significant variations in the brain distributions of each ErbB1 ligand. Among these ErbB1 ligands, HB-EGF protein levels were higher than those of TGFα and those of EGF were the lowest. TGFα protein was relatively enriched in the midbrain regions, while HB-EGF levels were most abundant in the cerebellum. Protein distributions of the EGF family members were discordant with previously reported mRNA distributions. In addition, there was significant basal ErbB1 phosphorylation detected with the largest amount of activation in the midbrain. These observations suggest that the activation of brain ErbB1 involves post-translational regulation of multiple EGF family members in a region-specific manner.

Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation.

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.

What's new in urology.

INFECTIOUS DISEASE AND CHRONIC PELVIC PAIN SYNDROME Interstitial cystitis, urethral syndrome, chronic pelvic pain syndrome, nonbacterial prostatitis, prostatodynia, and postejaculatory pain might all be different presentations of a single disease. The prevalence of these disease entities has an impact on the physician not only in the direct treatment of the diseases, but also in their relationship to other issues. A major issue for the urologist is the patient with the rising or elevated PSA, negative biopsies, and findings or symptoms compatible with prostatitis or chronic pelvic pain syndrome. A multicenter study was convened to determine if PSA or percent free PSA could be used as a marker for chronic pelvic pain syndrome or prostatitis. Another issue is whether PSA and percent free PSA values will be in the range associated with prostate cancer. In a study examining 424 patients with chronic prostatitis, etc, PSA values were compared with those from 114 age-matched asymptomatic controls. Findings showed total PSA in general was slightly higher in patients with chronic pelvic pain syndrome or prostatitis than in controls. But this slight elevation in level was not clinically significant, and although percent free PSA levels correlated with inflammation, the negative association was weak and not clinically significant. So the recommendation of the study was that male patients with chronic pelvic pain syndrome, etc, and abnormal PSA values should be evaluated for prostate cancer in accordance with current standard protocols. A study from France sought to determine the overlap between chronic pelvic pain syndrome and interstitial cystitis. Interstitial cystitis, per se, is poorly understood and is thought to predominate in women. It was noticed that a large percentage of men, when cystoscoped for their symptoms of nonbacterial prostatitis and prostatodynia, often have the cystoscopic appearance of interstitial cystitis by NIH-NIDDK criteria. This obviously raises the possibility that, in men, interstitial cystitis masquerades as nonbacterial prostatitis/prostatodynia or chronic pelvic pain syndrome. In this study, 300 men were identified who were diagnosed with chronic pelvic pain syndrome and were examined with cystoscopy and hydrodistension; 80% were found to have glomerulations that were believed to confirm the diagnosis of interstitial cystitis. Additionally, levels of nerve growth factors, tryptase, heparinbinding epidermal growth factor, and epidermal growth factor were evaluated in postprostatic massage urine and compared with these levels in controls. In these symptomatic young men, levels of nerve growth factor and tryptase were significantly elevated in those patients versus controls; heparin-binding epidermal growth factor levels were lower. All of these findings were statistically highly significant, raising the possibility that nerve growth factor, heparin-binding epidermal growth factor, and tryptase could serve as new markers for the evaluation of such patients. At the University of Pittsburgh, interesting research seeks to determine if gene therapy could be potentially useful for the treatment of interstitial cystitis patients. The investigators proposed that targeted and localized expression of endogenous opioid peptide in the bladder could be useful in treating bladder pain associated with interstitial cystitis. In one report from that institution, they described a gene gun method for the transfer of precursor molecules of endogenous opioid peptide in vivo and sought to investigate its therapeutic effect in rats using an acetic acid-induced bladder hyperactivity model. They found that, indeed, the proopiomelanocortin gene can be transferred to the bladder of these rats using the gene gun and the increased secretion of endorphin seemed to suppress bladder pain in the model. In a second study, they investigated the effects of enkephalin gene therapy using a replication deficient herpes simReceived June 11, 2003, Accepted June 12, 2003. From the Department of Urology, Eastern Virginia Medical School, Norfolk, VA. Correspondence address: Gerald H Jordan, MD, FACS, FAAP, Suite 100, 400 W Brambleton Ave, Norfolk, VA 23510.

The mitogenic potential of heparin-binding epidermal growth factor in the human endometrium is mediated by the epidermal growth factor receptor and is modulated by tumor necrosis factor-alpha.

Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFalpha has a cooperative effect on HB-EGF, EGF, TGFalpha, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFalpha in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFalpha in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFalpha, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation.

Altered expression of epidermal growth factor receptor ligands in tumor promoter–treated mouse epidermis and in primary mouse skin tumors induced by an initiation‐promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor α (TGFα), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFα mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands. Mol. Carcinog. 22:73–83, 1998. © 1998 Wiley-Liss, Inc.

Altered expression of epidermal growth factor receptor ligands in tumor promoter–treated mouse epidermis and in primary mouse skin tumors induced by an initiation-promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Mediators of estradiol-stimulated mitosis in the rat uterine luminal epithelium.

The effects of estradiol treatment, which stimulates cell division in rat uterine epithelial cells, on the in vivo expression of heparin-binding epidermal growth factor (HB-EGF), cyclin D1, and cyclin B1 messenger RNA (mRNA) in these cells have been examined using ribonuclease protection assays. Estradiol gave rise to significant increases in steady state levels of HB-EGF 2 and 24 h after treatment. Cyclin D1 mRNA levels were elevated 8 and 10 h after estradiol administration, corresponding to the G1 phase of the mitotic cycle, and cyclin B1 mRNA was only expressed 16-24 h after estradiol treatment, which corresponds to the G2 and M phases of the rat uterine epithelial cell cycle. Estradiol-stimulated increases in HB-EGF mRNA were not affected by treatment with cycloheximide, but were inhibited by the estrogen antagonist compound, ICI 164,384, demonstrating that the estrogen-stimulated increase in HB-EGF mRNA is a primary, estrogen receptor-mediated response of rat uterine epithelium to estradiol. Progesterone treatment, which blocks epithelial cells in G1 of the cycle, suppressed levels of HB-EGF mRNA below those observed in ovariectomized rats. These results indicate that HB-EGF mediates the regulatory effects of both estradiol and progesterone on rat uterine epithelial cell proliferation through an effect on the production of G1 phase molecules such as cyclin D1.