Heparin-binding Epidermal Growth Factor-like Growth Factor mRNA Expression Research Articles

HB-EGF expression as a potential biomarker of acquired middle ear cholesteatoma

Conclusions: The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma.Objective: To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation.Methods: A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed.Results: On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Abstract 1182: Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma

Abstract Neuroblastoma is the most common cancer in infancy. Current therapies are only modestly effective; patients with high-risk disease have less than a 50% chance of survival. High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble proteins, including heparan sulfate proteoglycans (HSPGs), which act to promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent pro-differentiating factor in neuroblastoma. Using microarray analysis, HBEGF mRNA expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from high-stage disease having decreased stroma and HBEGF. Soluble HBEGF increased neuroblast differentiation and decreased proliferation, while HBEGF knockdown decreased neuroblast differentiation. In patient samples, HBEGF expression correlates with SOX10, a neural crest differentiation marker, and the cell cycle inhibitor, CDKN1A. Alternatively, HBEGF negatively correlates with ASCL1, a primitive neuroectodermal marker, and the cell cycle promoter CCND1. HSPGs, including TβRIII, GPC1, GPC3, and SDC3 further promote the differentiating effects of HBEGF treatment by forming a complex with the epidermal growth factor receptor (EGFR) via glycosaminoglycan modifications, leading to activation of the Erk1/2 pathway and upregulation of the inhibitor of DNA binding transcription factor, whose expression correlates with HBEGF in patient samples. Inhibition of EGFR with erlotinib, lapatinib, and gefitinib diminish HBEGF-induced differentiation. These data identify a novel member of the pro-differentiating secretome and support the use of heparan sulfate mimetics in neuroblastoma, while cautioning against the use of EGFR inhibitors for neuroblastoma treatment. Citation Format: Angela L. Gaviglio, Gerard C. Blobe. Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1182.

Expression of Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) in Human Renal Cell Carcinoma

Background: The levels of expression of Heparin binding-epidermal growth factor like growth factor (HB-EGF) mRNA in tumor tissues and normal tissues of the excised kidney were compared in order to clarify the role of HBEGF inrenal cell carcinoma (RCC) derived from the proximal tubule. Method: Normal and tumor tissues were collected from surgical specimens of 16 cases pathologically diagnosed with RCC. Total RNA was extracted from these samples, and the level of expression of HB-EGF mRNA was measured by real-time quantitative PCR using a TaqMan probe after reverse transcription. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal standard. The expression levels of HB-EGF mRNA in normal and tumor tissues of the same case were compared, and statistical analysis was performed to evaluate the association between the expression level and various clinical-pathological factors in RCC. Results: Expression of HB-EGF mRNA was detected in 82% (13/16) of the normal tissues and 63% (10/16) of the tumor tissues. The expression level in the normal tissues was significantly 7-fold higher than that in the tumor tissues. No significant association was detected between the expression of HB-EGF mRNA and the clinical stage or prognosis of RCC. However, the pathological findings indicated that negative expression of ratio of HB-EGF was higher in RCC with more-advanced malignant progression. Conclusion: Our results indicated that it was unlikely that HB-EGF might play a role in determining the aggressiveness or clinical features in RCC. However, the decreased expression of HB-EGF mRNA in RCC tissues indicates that tumorigenesis of RCC may disrupt the normal regulatory system of HB-EGF.

HB-EGF but Not Amphiregulin or Their Receptors HER1 and HER4 Is Altered in Endometrium of Women With Unexplained Infertility

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors (HER1 and HER4) play a role in the human implantation process. Amphiregulin is a member of the EGF family but with unknown function in human fertility. It has been suggested that some women with unexplained infertility have defective endometrial development. The aim of this study is to determine the presence of amphiregulin and the receptors HER1 and HER4 in normal human endometrium throughout the menstrual cycle. In addition, the present study aims to compare endometrium from women with unexplained infertility with endometrium from women with male factor infertility and healthy fertile controls. Immunohistochemistry and real-time polymerase chain reaction were used to determine the expression of HB-EGF, HER1, HER4, and amphiregulin. The stromal staining of HER1 and the epithelial staining of HER4 were most intense in the mid- and late-secretory-phase endometrium. Amphiregulin did not vary during the menstrual cycle. In the mid-secretory phase, the protein expression of HB-EGF was lower in endometrium from women with unexplained infertility versus normal endometrium and endometrium from women with male factor infertility. HB-EGF and HER4 mRNA expression in mid-secretory endometrium of women with unexplained and male factor infertility were increased compared with normal controls. Impaired endometrial expression of certain members of the EGF family may contribute to infertility in some women with unexplained infertility.

The Effect of Heparin Binding Epidermal Growth Factor on the Hypertrophy / Hyperplasia of Human Bladder Smooth Muscle

Puropose: The bladder outlet obstruction (BOO) causes a bladder hypertrophy and/or hyperplasia with time. Heparin binding epidermal growth factor like growth factor (HB-EGF) is a potent mitogen for bladder smooth muscle cell (BMSC), causing significant cellular hypertrophy. Few studies have evaluated the effect of HB-EGF on bladder hypertrophy or hyperplasia. We studied to evaluated the effect of HB-EGF on the hypertrophy or hyperplasia of human BSMC and fibroblast. Material and methods: To evaluate the change of expression of HB-EGF mRNA of human BMSC under a constant stimuli of 40cm-H20 pressure during 0, 1, 3 and 5 hours, RT-PCR was perfromed. The human BMSC and bladder fibroblasts with the addition of the recombinant HB-EGF 25ng/ml were performed H-thymidine and H-leucine incorporation assay for DNA and protein production, respectively. Results: The expression of HB-EGF mRNA in BMSC under a constant stimuli of 40cm-H20 pressure was up-regulated with time (p<0.05). The optimal concentration of HB-EGF was 25ng/ml. The H-Lecine and H-thymidine activity in both human BSMC and fibroblast were significantly increased in addition of 25ng/ml of HB-EGF (p<0.05 and p<0.05, respectively). Conclusions: Constant hydrostatic pressure induces the expression of HB-EGF in human BSMC. Subsequently HB-EGF induces hypertrophy and hyperplasia of human BSMC and fibroblast in vitro. Our data support that HB-EGF is one of relevant BOO-induced growth factors. (J Korean Continence Soc 2008;12:169-77)

Synergistic effects of acitretin and narrow-band UVB on inducing the expression of heparin-binding epidermal-growth-factor-like growth factor in normal human keratinocytes

It has been reported that heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) plays an important role in regulating keratinocyte growth after retinoic acid treatment. In clinic, there are synergistic effects of retinoic acid plus ultraviolet (UVB) on improving efficacy and reducing the side effects in treating psoriasis, while the mechanism is not fully clear. Therefore, this study was taken to examine the alteration of cell proliferation and HB-EGF expression in cultured normal human keratinocytes after acitretin and/or narrow-band UVB (NB-UVB) exposure. After 12 h incubation with acitretin (0.1-1 micromol/L) and/or following NB-UVB (50-100 mJ/cm2) irradiation in normal human keratinocytes, the proliferation potency of the cells was evaluated by MTT colorimetric assay, and HB-EGF protein and mRNA was detected by cytoimmunochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results revealed 1 micromol/L acitretin combined with 100 mJ/cm2 NB-UVB showed strongest inhibition of keratinocyte proliferation and highest induction of HB-EGF mRNA expression (19.7% and 8.6-fold, respectively), the others were 1 micromol/L acitretin (14.4%, 7.1-fold), 0.1 micromol/L acitretin (10.2%, 3.2-fold), 100 mJ/cm2 NB-UVB (8.7%, 2.5-fold) and 50 mJ/cm2 NB-UVB (5.4%, 1.8-fold). Pattern of HB-EGF protein expression was similar to mRNA expression in keratinocytes upon above cultural conditions. Our results suggest acitretin or NB-UVB single treatment can dose-dependently inhibit keratinocyte proliferation and induce HB-EGF expression and there are synergistic effects when they are combined.

Gonadotropin Stimulation May Not Affect the Expression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Murine Endometrium

Background: Present human data reveal that gonadotropin stimulation for in vitro fertilization and embryo transfer (IVF-ET) is detrimental to uterine receptivity. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is important in the early stage of mouse embryonic implantation since it is expressed in the murine endometrium before the attachment reaction. Gonadotropin receptors have been found on the murine endometrium. In this study, we assessed the expression of HB-EGF mRNA on the gonadotropin-stimulated murine endometrium and sought evidence of whether the gonadotropin-induced, altered uterine receptivity is associated with a change in the HB-EGF expression. Methods: Quantitative, competitive reverse-transcription polymerase chain reaction (PCR) and immunohistochemistry were performed to evaluate the expression of HB-EGF mRNA on the stimulated murine endometrium from pregnant uteri of gestation day (g.d.) 5.0 after injection of saline, or pregnant mare serum gonadotropin (PMSG) 5 or 10 IU. Results: The expression of HB-EGF mRNA showed no significant increase or decrease in the endometrial expression in PMSG groups, compared to the control group (natural coitus with saline injection), on g.d. 5.0. This finding was consistent with the immunostaining of HB-EGF protein, and there was no significant difference in the staining intensity among the 3 groups. Conclusion: This study reveals that ovarian stimulation by gonadotropin may not influence the expression of HB-EGF in the murine endometrium. Further investigations are necessary to elucidate whether embryonic HB-EGF receptor status relative to endometrial HB-EGF expression is affected by ovarian stimulation.

Growth and stretch response of human exstrophy bladder smooth muscle cells: molecular evidence of normal intrinsic function

To establish primary cultures of smooth muscle cells (SMC) from human exstrophic bladders (E-SMC), and determine their in vitro growth dynamics and responses to mechanical stretch. Primary cultures of E-SMC from three patients were established from exstrophic bladder tissue using an explant method. Growth dynamics were assessed using tetrazolium-dye uptake. The DNA synthesis rate in response to cyclic stretch-relaxation was determined with thymidine-incorporation assays. Expression of the SMC mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in response to mechanical stretch was determined using semiquantitative reverse transcription-polymerase chain reaction. The approximate doubling time of the E-SMC grown in the presence of serum was 4 days, consistent with growth rates of SMC reported previously. E-SMC exposed to stretch had greater DNA synthesis, albeit to a lesser extent than previously seen with non-exstrophic SMC. The expression of HB-EGF was also increased in cells exposed to mechanical stimuli, consistent with our previous finding of stretch-regulated HB-EGF gene expression in bladder SMC. E-SMC had growth characteristics similar to those previously reported in non-exstrophic cells. E-SMC also had stretch-induced expression of HB-EGF mRNA. These observations provide evidence that despite development in an abnormal defunctionalized state, E-SMC retain the potential for normal growth, and may modulate this response through mechanisms similar to those operating in normal bladder SMC.

Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5 mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.

Differential expression of heparin-binding EGF-like growth factor (HB-EGF) mRNA in normal human keratinocytes induced by a variety of natural and synthetic retinoids.

It was recently revealed that epidermal growth following topical treatment with all-trans retinoic acid (atRA) was at least partly induced by heparin-binding epidermal growth factor-like growth factor (HB-EGF) released from suprabasal keratinocytes. Since proliferation of keratinocytes appears to be one of the critical roles of atRA in depigmentation treatment and promotion of wound healing, HB-EGF is considered suitable for assessing the therapeutic value of topical retinoids. In this study, HB-EGF mRNA expression in normal human keratinocytes after atRA treatment was examined, and the effects of a variety of natural and synthetic retinoids were compared. The results of reverse transcription polymerase chain reaction (RT-PCR) suggested that induction of differentiation increased HB-EGF mRNA expression in cultured keratinocytes. Real-time PCR analyses revealed that HB-EGF mRNA expression was elevated dose-dependently with atRA, peaking at 12 h. This elevation was more prominent in confluent keratinocytes than in subconfluent cells, suggesting that differentiated keratinocytes are more subject to stimulation of HB-EGF expression by atRA than proliferating keratinocytes. HB-EGF mRNA was upregulated in differentiation-induced keratinocytes by all retinoids used in this study at 1 micromol/l, and marked upregulation was seen when treated with three isotypes of retinoic acid (atRA, and 9-cis and 13-cis retinoic acid). RARalpha-selective agonists (Am80, Am580, ER-38925, and TAC-101) and a panagonist of RARs (Re80) caused relatively low elevation of HB-EGF transcripts, as did all-trans retinol (Rol) and all-trans retinal (Ral). Although another panagonist (Ch55) showed the highest elevation of HB-EGF mRNA, it was relatively cytotoxic at the concentration employed. Ral and Rol were found to upregulate HB-EGF when used at 100 micromol/l to 1 mmol/l, to a similar extent of atRA at 1-10 micromol/l. The capacity of retinoids to upregulate HB-EGF may be an important index for investigation and development of an ideal synthetic retinoid, which has maximum benefits and minimum side-effects

Vanadium-induced HB-EGF expression in human lung fibroblasts is oxidant dependent and requires MAP kinases

Vanadium pentoxide (V(2)O(5)) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H(2)O(2)) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V(2)O(5) treatment. V(2)O(5)-induced HB-EGF mRNA expression was abolished by N-acetyl-l-cysteine, suggesting an oxidant-mediated effect. Exogenous H(2)O(2) (>10 microM) mimicked the effect of V(2)O(5) in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H(2)O(2) (1-2 microM), and the addition of V(2)O(5) depleted the endogenous H(2)O(2) pool within minutes. V(2)O(5) caused a subsequent increase of H(2)O(2) into the culture medium at 12 h. However, the burst of V(2)O(5)-induced H(2)O(2) occurred after V(2)O(5)-induced HB-EGF mRNA expression at 3 h, indicating that the V(2)O(5)-stimulated H(2)O(2) burst did not mediate HB-EGF expression. Either V(2)O(5) or H(2)O(2) activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V(2)O(5)- or H(2)O(2)-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H(2)O(2) in V(2)O(5) treated cells, yet the action of V(2)O(5) in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V(2)O(5) with endogenous H(2)O(2).

Pre-eclampsia and expression of heparin-binding EGF-like growth factor

Pre-eclampsia is a disorder of pregnancy associated with poor extravillous cytotrophoblast invasion and above-normal rates of apoptosis in the trophoblast. Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) has strong cytoprotective activity and is an important signalling protein that regulates trophoblast invasion during early placentation. We aimed to establish whether HB-EGF expression is altered in placentae of pre-eclamptic women. We assessed the expression of HB-EGF mRNA and protein by in-situ hybridisation and immunohistochemical techniques, respectively, in archived placental tissues from pregnancies terminated at around 20 weeks of gestation, and from women delivering between weeks 19 and 35 of gestation with preterm labour, small for gestational age infants, or pre-eclampsia. HB-EGF mRNA and protein were expressed in villous and extravillous cytotrophoblast cells up to week 35 of gestation in placentae from women who delivered preterm. Similar levels of HB-EGF protein were found in the placentae of women who were not in labour. HB-EGF expression was reduced about five-fold (p=0.0001) in pre-eclamptic pregnancies. Fetal growth retardation, which has been linked with shallow trophoblast invasion and moderate apoptosis, was associated with placentae expressing intermediate levels of HB-EGF. In pre-eclampsia, deficient HB-EGF signalling during placental development could impair trophoblast survival, differentiation, and invasion, leading to poor placental perfusion and hypertension.

Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching

Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching. The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis. Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media. Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoelectron microscopy revealed that the initial recovery of cellular proHB-EGF was localized predominantly to areas of cell-cell attachment. No tubule-like structures were observed in similarly treated NRK52E cells transfected with the vector alone. In collagen gels, NRKproHB-EGF developed short tubule-like structures in the absence of TPA treatment, but with simultaneous TPA treatment, longer and more arborized structures developed. MMP-1 mRNA and immunoreactive protein increased in the TPA-treated cells, suggesting that protein kinase C-mediated collagenase activity was important for the observed tubulogenesis. However, inhibition of EGF receptor tyrosine kinase with AG 1478 significantly blunted the TPA-induced tubulogenesis by NRKproHB-EGF grown in collagen gels. These results indicate that membrane-bound HB-EGF can mediate both epithelial cell branching and cell motility. Localization of proHB-EGF to the site of cell-cell contact and development of tubule-like structures in collagen gels suggests that proHB-EGF may be an important morphogen for renal epithelial cells.

Concomitant expression of heparin-binding epidermal growth factor-like growth factor mRNA and basic fibroblast growth factor mRNA in myocardial infarction in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is mitogenic and chemotactic for many cell types. HB-EGF is induced in pathological states which require cell mitogenesis and proliferation, including angiogenesis, and has been reported to interact functionally with basic fibroblast growth factor (bFGF). To test our hypothesis that HB-EGF mRNA expression is increased in myocardial infarction, we used Northern hybridization in rats to investigate the expression of HB-EGF and EGF receptor mRNAs expression in the infarct zone compared to the expression of bFGF and FGF receptor mRNAs. We also performed in situ hybridization to identify the cells responsible for HB-EGF mRNA production. HB-EGF mRNA rapidly increased after ligation (mean +/- SE, 5.6+/-0.23-fold increase at 6 hours compared to the preligation heart levels) and reached a maximum level (9.1+/-0.42-fold increase) around 12 hours. HB-EGF mRNA then gradually decreased on day 1 (5.8+/-1.0-fold increase), day 2 (3.2+/-0.94-fold increase) and day 3 (1.9+/-0.33-fold increase) after ligation. Parallel changes in bFGF mRNA expression were observed (6, 12 hours, days 1, 2 and 3; 3.6+/-0.42-, 5.3+/-0.12-, 2.3+/-0.12-, 1.7+/-0.03- and 0.95+/-0.03-fold increase, respectively). EGF receptor (ErbB-1) mRNA was gradually increased on day 2 (2.4+/-0.53-fold increase), day 7(4.0+/-0.61-fold increase) and day 14 (7.0+/-0.61-fold increase). Similarly, FGF receptor (FGF receptor-1) mRNA was gradually increased (days 2,7 and 14; 1.3+/-0.13-, 1.5+/-0.17- and 2.3+/-0.15-fold increase, respectively). Reperfusion after a 2-hour ligation (too late to salvage myocytes) enhanced HB-EGF (12 hours, 16.8+/-1.8-fold increase) and bFGF (12 hours, 10.4+/-1.1-fold increase) mRNA expression. The cells responsible for the increased production of HB-EGF mRNA were shown by in situ hybridization to be surviving myocytes located in the infarct peripheral zone around infarct necrotizing tissue. In conclusion, our results demonstrated a rapid increase in HB-EGF mRNA expression concomitant with an increase in bFGF mRNA expression, suggesting that HB-EGF and bFGF might play some role in the course of pathological changes in the infarct in the early inflammatory phase. Reperfusion at times too late to salvage myocytes accelerated sequential changes in the expression of both HB-EGF and bFGF mRNAs.

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor: a mitogen for lung fibroblasts.

The bronchial epithelium is a potential source of growth factors that could mediate airway fibrosis during the progression of diseases such as asthma and chronic bronchitis. We report that conditioned medium (CM) from normal human bronchial epithelial cells (NHBECs) contains mitogenic activity for human lung fibroblasts that is blocked by the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478 and by neutralizing antibodies raised against heparin-binding epidermal growth factor-like growth factor (HB-EGF). Neutralizing antibodies against other EGF-R ligands (EGF and transforming growth factor-alpha) or other antibodies against growth factors (platelet-derived growth factors, insulin-like growth factor-1) had no affect on the mitogenic activity of NHBEC-CM. HB-EGF messenger RNA (mRNA) expression in NHBEC was detected by reverse transcriptase/polymerase chain reaction and Northern blot analysis. HB-EGF protein was detected by enzyme-linked immunosorbent assay. Vanadium pentoxide (V2O5), a fibrogenic metal associated with occupational asthma, caused a several-fold increase in HB-EGF mRNA expression and protein, whereas the inert metal titanium dioxide had no effect on HB-EGF expression. V2O5-induced HB-EGF mRNA expression was inhibited by the EGF-R tyrosine kinase inhibitor AG1478, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and the MAP kinase kinase inhibitor PD98059. Finally, HB-EGF induced the production of fibroblast growth factor (FGF)-2 by human lung fibroblasts and anti-FGF-2 antibody partially blocked the mitogenic activity of NHBEC-CM on fibroblasts. These data suggest that HB-EGF is a fibroblast mitogen produced by NHBECs and that induction of an FGF-2 autocrine loop in fibroblasts by HB-EGF accounts for part of this mitogenic activity.

Oxidant Stress Activates AP-1 and Heparin-Binding Epidermal Growth Factor-Like Growth Factor Transcription in Renal Epithelial Cells

Ischemia/reperfusion injury increases the expression of bioactive heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the rat kidney, suggesting that oxidant stress or cell injury related to oxidant stress might affect HB-EGF expression in the injured renal parenchyma. We utilized a nontransformed rat renal epithelial cell line (NRK-52E cells) to investigate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E cells, and at concentrations that induced sublethal cell injury, hydrogen peroxide (H₂O₂) increased HB-EGF mRNA expression 4.7-fold. The free radical scavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA induction. In contrast, another free radical scavenger, pyrrolidine thiocarbamate (PDTC), augmented H₂O₂-mediated HB-EGF expression. Since PDTC has been reported to augment AP-1-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H₂O₂ administration to NRK-52E cells did increase nuclear extract DNA-binding activity to a consensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,000 bp of the human HB-EGF 5′-untranslated region, we determined that H₂O₂ administration increased CAT activity 5.5-fold. Truncation or deletion mutations of a putative AP-1-binding site reduced the H₂O₂-stimulated activity by >60%, and there was increased DNA binding of nuclear extracts from H₂O₂-treated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti-fos and jun antibodies inhibited this binding, and there was no binding to an oligonucleotide in which the putative AP-1 site was mutated.The site of the residual activation was found to exist in the most proximal 5′-untranslated region (–121 to +60), which contains two putative SP1 sites. Timing and localization of AP-1-binding activity from nuclear extracts from the post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, which appears to be mediated in part by an increase in AP-1 binding. This activation may play an important role in the induction of HB-EGF mRNA in response to tissue injury and may regulate early stages of recovery following ischemic damage.

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.