Heparin-binding Epidermal Growth Factor-like Growth Factor mRNA 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.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

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.

Forebrain specific heparin-binding epidermal growth factor-like growth factor knockout mice show exacerbated ischemia and reperfusion injury

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hypoxia-inducible neuroprotective protein that also stimulates proliferation of neuronal precursor cells. In this study, we investigated the possible role of HB-EGF in ischemia and reperfusion injury by measuring the changes in its mRNA expression following focal cerebral ischemia. We also examined neural damage after a middle cerebral artery occlusion (MCAO) and reperfusion in ventral forebrain specific HB-EGF knockout (KO) mice. The levels of HB-EGF mRNA in the cerebral cortex of wild-type (WT) mice were significantly increased 3-24 h after MCAO and reperfusion. Cerebral infraction in HB-EGF KO mice was aggravated at 1 day and 6 days after MCAO and reperfusion compared with WT mice. The number of terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) and an oxidative stress marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG) positive cells, were higher in HB-EGF KO mice than in WT mice. On the other hand, fewer bromodeoxyuridine (BrdU) positive cells were found in the subventricular zone in HB-EGF KO mice compared with WT mice. These results indicate that HB-EGF may play a pivotal role in ischemia and reperfusion injury and that endogenously synthesized HB-EGF is necessary for both the neuroprotective effect and for regulation of cell proliferation in the subventricular zone.

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.

The chemotherapeutic agent VP16 increases the stability of HB-EGF mRNA by a mechanism involving the 3′-UTR

VP16 is a chemotherapeutic agent that introduces DNA damage. We demonstrate that cellular stress induced by VP16 in the human cervix cancer cell line HeLa increases the HB-EGF (heparin binding epidermal growth factor like growth factor) mRNA level dose dependently. Maximal induction (10-fold) was observed at 20–40 μM VP16. Increased HB-EGF peptide levels accompanied the increase in HB-EGF mRNA. We investigated the molecular mechanism involved in HB-EGF mRNA induction by VP16. Transcription was only slightly increased (60%) as determined by real-time PCR quantification of transcription from a reporter plasmid containing the HB-EGF promoter in front of the luciferase gene. In contrast, HB-EGF mRNA stability was increased significantly by VP16 as demonstrated by monitoring HB-EGF mRNA decay in cells treated with the transcriptional inhibitor actinomycin D. The 3′-UTR (3′-untranslated region) of HB-EGF was inserted at the 3′-end of LacZ mRNA. VP16 treatment of the cells caused a 5-fold increase in this chimeric mRNA, as compared to LacZ without this 3′-UTR. A 186 nucleotide region of HB-EGF contains five of the six AUUUA sequences found in the 1454 nucleotide 3′-UTR of HB-EGF and we demonstrate that this region caused an approximately 3-fold induction of LacZ mRNA when inserted at the 3′-end, as compared to LacZ without any insertion at the 3′-end, demonstrating that a significant proportion of the effect resides in this region. Induction of HB-EGF by VP16 has important implications as HB-EGF has been reported to prevent cell death, which might lower the efficacy of chemotherapy. We demonstrate that mRNA stability and in particular the HB-EGF 3′-UTR is involved in the HB-EGF mRNA induction.

Gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor and human epidermal growth factor receptors in human corpus luteum

The objective of this study was to elucidate gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and human epidermal growth factor receptor (HER) family in the human ovary during luteal growth and regression. Ovaries obtained from pre-menopausal women were used for immunohistochemistry and semiquantitative RT-PCR analysis. Immunoreactive HB-EGF was not detected in follicles or oocyte, while HB-EGF became apparent in granulosa luteal cells in the early luteal phase, and most abundant in the mid-luteal phase, but less abundant in the late luteal phase. Immunostaining for HER1 was very weak in granulosa luteal cells in the early and mid-luteal phases, and was not detected in the late luteal phase. Immunoreactive HER4 was abundant in the early luteal phase and became less abundant in the mid-luteal phase, whereas it was negative in the late luteal phase. Semiquantitative RT-PCR analysis revealed that HB-EGF and HER1 mRNA levels were high in the mid-luteal phase, whereas HER4 mRNA expression was high in the early luteal phase. HB-EGF may play a vital role in regulating luteal growth in a juxtacrine manner and through activating HER4 signalling.

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.

17β-Estradiol enhances heparin-binding epidermal growth factor-like growth factor production in human keratinocytes

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) enhances reepithelialization in wounds. Estrogen is known to promote cutaneous wound repair. We examined the in vitro effects of 17beta-estradiol (E2) on HB-EGF production by human keratinocytes. E2 or membrane-impermeable BSA-conjugated E2 (E2-BSA) increased HB-EGF secretion, mRNA level, and promoter activity in keratinocytes. E2 or E2-BSA enhanced in vitro wound closure in keratinocytes, and the closure was suppressed by anti-HB-EGF antibody. Activator protein-1 (AP-1) and specificity protein 1 (Sp1) sites on HB-EGF promoter were responsible for the E2- or E2-BSA-induced transactivation. Antisense oligonucleotides against c-Fos, c-Jun, and Sp1 blocked E2- or E2-BSA-induced HB-EGF transactivation. E2 or E2-BSA enhanced DNA binding and transcriptional activity of AP-1 and generated c-Fos/c-Jun heterodimers by inducing c-Fos expression. E2 or E2-BSA enhanced DNA binding and transcriptional activity of Sp1 in parallel with the enhancement of Sp1 phosphorylation. These effects of E2 or E2-BSA were not blocked by the nuclear estrogen receptor antagonist ICI-182,780 or anti-estrogen receptor-alpha or -beta antibodies but were blocked by inhibitors of G protein, phosphatidylinositol-specific PLC, PKC-alpha, and MEK1. These results suggest that E2 or E2-BSA may enhance HB-EGF production via activation of AP-1 and Sp1. These effects of E2 or E2-BSA may be dependent on membrane G protein-coupled receptors different from nuclear estrogen receptors and on the receptor-mediated activities of phosphatidylinositol-specific PLC, PKC-alpha, and MEK1. E2 may enhance wound reepithelialization by promoting HB-EGF production in keratinocytes.

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.

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT–PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT–PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.

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

The Role of Ultraviolet Irradiation and Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Pathogenesis of Pterygium

Ultraviolet (UV) light is one of the major factors implicated in the pathogenesis of pterygium. The mechanism by which UV light induces this disease remains elusive. The aim of this study was to evaluate the effects of UVB irradiation on the expression of growth factors in cultured pterygium epithelial cells and to demonstrate their distribution within pterygium. We cultured pterygial epithelial cells from pterygium explants and these cells were exposed to 20 mJ/cm(2) of UVB. Total RNA was extracted at 0, 6, and 12 hours after irradiation. (32)P-labeled cDNA was synthesized and analyzed using microarray technology to determine the differential expression of 268 growth factor and cytokine related genes. Semiquantitative reverse transcriptase-polymerase chain reaction was used to corroborate this data. Conditioned media derived from cells exposed to UVB irradiation was analyzed for protein expression by enzyme-linked immunosorbent assay. Immunohistochemistry was used to evaluate the distribution of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pterygium tissue. Analysis of the hybridization signals revealed that the genes encoding HB-EGF, fibroblast growth factor 3, and cytotoxic trail ligand receptor were consistently elevated at 6 and 12 hours after UVB treatment. HB-EGF mRNA was elevated 6.8-fold at 6 hours after irradiation and was augmented in culture supernatants after the same treatment. Furthermore, HB-EGF reactivity was identified in the epithelium and vasculature of pterygium by immunohistochemistry. HB-EGF was present in normal limbal epithelium, although it was not induced in cultured limbal epithelial cells by UV irradiation. HB-EGF is a potent mitogen, localized in pterygium tissue, and significantly induced by UVB in pterygium-derived epithelial cells. We postulate that this growth factor is a major driving force in the development of pterygia and a means by which UV irradiation causes the pathogenesis of pterygium.

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).

Alteration and Significance of Heparin-Binding Epidermal-Growth-Factor-Like Growth Factor in Psoriatic Epidermis

Background: Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) has proved to be a mitogen of keratinocytes, which may be involved in the pathogenesis of inflammatory diseases, but its mechanism in psoriasis remains unknown. Objective: To investigate the alteration of HB-EGF in the epidermis of active psoriasis vulgaris. Methods: The expression of HB-EGF in normal skin tissues, uninvolved tissues and psoriatic lesions was detected by in situ hybridization and immunohistochemistry. Results: HB-EGF mRNA and protein were located in the basal layer of normal epidermis (10/10, 100.00%), with nearly no expression in the suprabasal layers (1/10, 10.00%); the expression of HB-EGF was located not only in the basal layer (21/21, 100.00%), but was also seen as focal overexpression in the suprabasal layers of uninvolved epidermis (20/21, 95.24%) and the marginal part of psoriatic lesions (18/21, 85.71%), while nearly no expression of HB-EGF was found in the central part of psoriatic lesions (1/21, 4.76%). Conclusion: HB-EGF may play an important role in the early pathogenesis of psoriasis.