Endopeptidase Expression Research Articles

Serum level of substance P and stress in acne-vulgaris patients

ObjectiveThe aim of the study was to evaluate the relationship between stress and serum concentration of substance P (SP) in acne-vulgaris patients.BackgroundAcne vulgaris is a multifactorial disorder of the pilosebaceous unit. The clinical picture can vary significantly, from mild comedonal acne to fulminant systemic disease. Although all age groups may be affected by its many variants, the peak incidence is during adolescence. Various studies have proved that neuropeptides affect the activity of the pilosebaceous unit by stimulating proliferation and differentiation of sebaceous glands, lipid synthesis, and induction of neutral endopeptidase expression in sebaceous cells, especially SP.Patients and methodsA case–control study included 40 patients with acne vulgaris and 40 age-matched and sex-matched controls. Emotional stress was analyzed with the use of Social Readjustment Rating Scale, whereas the methods of coping with stress were assessed with the Coping Inventory for Stressful Situation questionnaire, followed by taking blood samples to assess SP serum levels using the enzyme-linked immunosorbent-assay method.ResultsSP was significantly higher in the acne-patient group versus the control group (P < 0.05). A statistically significant difference was observed between acne patients and control groups regarding the Social Readjustment Rating Scale (P < 0.05).ConclusionThere was a statistically significant relation between stress scale and serum SP level in acne vulgaris.

Cadmium-induced toxicity increases prolyl endopeptidase (PREP) expression in the rat testis.

During the differentiation of the male gamete, there is a massive remodeling in the shape and architecture of all the cells of the seminiferous epithelium. The cytoskeleton, as well as many associated proteins with it, plays a pivotal role in this process. The testis is particularly susceptible to environmental pollutant, which can lead to injury and impairment of normal spermatozoa production. Cadmium (Cd) is one of the major chemical environmental toxicants in economically developed countries. Food and cigarettes are the main sources of exposure to this element. Here, the protective role of zinc (Zn) to prevent the testicular toxicity in male adult rats after prenatal and during lactation exposure to Cd has been assessed. Altered testicular histology at the interstitial and germinal levels was found, whereas Zn supply completely corrected Cd toxicity. Moreover, the effects of these metals on the testicular expression and localization of the protease prolyl endopeptidase (PREP) were evaluated. Interestingly, the results showed an increase of PREP messenger RNA and protein. Data were corroborated by immunofluorescence. This study raises the possibility of using PREP as a new fertility marker.

C-Type Natriuretic Peptide Dampens Fibroblast Growth Factor-23 Expression Through MAPK Signaling Pathway in Human Mesangial Cells

C-type natriuretic peptide (CNP) is believed to be produced locally in the kidneys and possess several renoprotective properties. In contrast, fibroblast growth factor (FGF) -23 elevates in the early stage of chronic kidney disease and predicts its outcomes. Currently, several studies have demonstrated that CNP and FGF-23 act through a close pathway, and moreover, FGF-23/mitogen-activated protein kinase (MAPK) can be obviously suppressed by CNP. In the present study, human mesangial cells (MCs) were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 h, respectively. CNP administration significantly suppresses MCs proliferation in a time- and dose-dependent manner. As a down-stream signaling of CNP activation, the expressions of natriuretic peptide receptor (NPR)-B, cyclic guanosine monophosphate-dependent protein kinases II and NPR-C were obviously augmented, whereas neutral endopeptidase expression was significantly decreased after CNP treatment in MCs. FGF-23, FGF receptor-1 and RAF-1 experienced a pronounced down-regulation in MCs with different doses of CNP throughout the whole observational period. CNP may dampen FGF-23 expression via MAPK signaling pathway in MCs.

Synergistic relationship between dipeptidyl peptidase IV and neutral endopeptidase expression and the combined prognostic significance in osteosarcoma patients

Neutral endopeptidase (NEP/CD10) and dipeptidyl peptidase IV (DPP IV/CD26) are both ubiquitous glycopeptidases which play important roles in tumor pathogenesis and development. The aim of this study was to investigate the expression patterns and the prognostic significance of CD10 and CD26 in osteosarcoma patients. CD10 and CD26 expression in 116 pairs of primary osteosarcoma and corresponding noncancerous bone tissue samples from the same specimens were detected by immunohistochemistry. The Spearman's correlation was calculated between the expression levels of CD10 and CD26 in osteosarcoma tissues. The associations of CD10 and CD26 expression with the clinicopathologic features and with the prognosis of osteosarcoma were subsequently assessed. Both CD10 expression and CD26 expression in osteosarcoma tissues were significantly higher than those in corresponding noncancerous bone tissue samples (both P < 0.001). Overexpression of CD10 and CD26 were respectively observed in 68.10 % (79/116) and 70.69 % (82/116) of osteosarcoma tissues. A significant correlation was found between CD10 expression and CD26 expression in osteosarcoma tissues (r = 0.83, P < 0.001). In addition, combined overexpression of CD10 and CD26 was observed in 52.59 % (61/116) of osteosarcoma tissues. CD10-high/CD26-high expression was significantly correlated with advanced clinical stage (P = 0.001), positive metastatic status (P = 0.001), shorter overall (P < 0.001) and disease-free (P < 0.001) survival in patients with osteosarcomas. Furthermore, multivariate survival analysis showed that clinical stage, metastatic status, CD10 expression, CD26 expression and combined expression of CD10/CD26 were all independent prognostic factors for predicting both overall and disease-free survival of osteosarcoma patients. Interestingly, combined expression of CD10/CD26 had a better prognostic value than other features. This retrospective study offer the convincing evidence for the first time that the overexpression of CD10 or CD26 may be an important feature of human osteosarcomas, and the combined expression of CD10/CD26 may be an efficient prognostic indicator for this disease.

Abstract 3023: Decreased neutral endopeptidase expression regulates the autocrine proliferative effects of substance P on cholangiocarcinoma growth

Abstract Cholangiocarcinoma (CCA) originates from the neoplastic transformation of cholangiocytes, which are the epithelial cells that line the bile ducts. CCAs are neuroendocrine tumors and secrete and respond to neuropeptides with alterations in cell proliferation via autocrine pathways. For example, the expression/secretion of neuroendocrine factors such as NPY and serotonin are dysregulated in cholangiocarcinoma, which exert local control on tumor cell proliferation. The neuropeptide substance P (SP) stimulates normal biliary hyperplasia by binding to the neurokinin-1 receptor (NK-1R) stimulating ERK1/2 activity. Neutral endopeptidase (NEP) is a metalloprotease that degrades a number of secreted neuropeptides including SP. The AIM of our study was to evaluate the role of NEP in the autocrine regulation of CCA growth by SP. METHODS: The intra- and extra-hepatic CCA lines, Mz-ChA-1, SG231, CCLP, HuCCT-1, HuH-28, and TFK-1, and non-malignant biliary line, H-69, were utilized. We evaluated NK1R, tachykinin 1 (TAC1, gene encoding SP) and NEP expression by qPCR, FACS and immunofluorescence in cell lines and by immunohistochemistry in commercially available CCA tissue arrays. The secretion of SP in all lines was measured by EIA. The dose-dependent effect of SP (10−6 to 10−11 M for 48 hrs) on cell growth and SP-induced (10−7 M) cell growth in the presence/absence of PD98059 (MEK1 inhibitor) was evaluated by MTS proliferation assay. The effect of SP on phosphorylation of ERK1/2 was determined by Western blot in the presence/absence of PD98059. To determine the role of NEP in the regulation of CCA growth, NEP was overexpressed in Mz-ChA-1 cells before evaluating: (i) NEP expression by qPCR and SP secretion by EIA kits; (ii) proliferation by MTS assay; and (iii) expression of Cyclin D1, PCNA and VEGF-A/C (factors regulating CCA growth). RESULTS: All CCA cell lines and human CCA tissue array samples were positive for NK-1R and TAC1. The CCA lines secreted higher levels of SP compared to H69. Correspondingly, NEP expression levels were significantly reduced in CCA lines compared to nonmalignant H69 cells and in human CCA tissue arrays compared to normal bile ducts. SP increased CCA growth (∼60% at 10−7 M), which was dependent upon ERK1/2 activity. Overexpression of NEP (∼80%, the SP degrading enzyme) in Mz-ChA-1 cells decreased SP secretion and significantly decreased cell growth, which was associated with decreased expression of Cyclin D1, PCNA and VEGF-A/C compared to control transfected Mz-ChA-1. CONCLUSIONS: SP is mitogenic for CCA regulating growth in an autocrine fashion that is associated with decreased NEP expression. Targeting the NEP/SP/NK1R axis may represent a novel therapeutic approach for cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3023. doi:1538-7445.AM2012-3023

Atrial fibrillation down-regulates renal neutral endopeptidase expression and induces profibrotic pathways in the kidney

Recent studies suggest that atrial fibrillation (AF) substantially influences microvascular flow in ventricular myocardium. This process may contribute to the occurrence of heart failure in AF. In general, development of heart failure and renal dysfunction go hand-in-hand causing systemic fluid overload and oedema. So far, it is unknown whether AF itself influences renal function. The aim of the present study was to determine the impact of AF on renal gene expression in a closed chest rapid atrial pacing model. A total of 14 pigs were studied. In five pigs, rapid atrial pacing (AT) was performed for 7 h (600 bpm); in five additional animals, rapid atrial pacing was performed in the presence of irbesartan infusion (irbesartan group). Four pigs were instrumented without interventions (sham). After the pacing period, renal expression of collagen I alpha 1 and I alpha 3, transforming growth factor-beta (TGF-beta), neutral endopeptidase (NEP; the main enzyme involved in natriuretic protein metabolism), and atrial natriuretic peptide (ANP) were determined by RT-PCR and immunoblot analysis. Functional in vitro experiments were performed using HEK-293 kidney cells. Renal mRNA expression of NEP was substantially down-regulated during AT (AT: 12.7 +/- 9.3% vs. sham: 100 +/- 43.4%; P < 0.01). Results at the mRNA level were confirmed at the protein level. Irbesartan therapy did not prevent down-regulation of NEP. In contrast, TGF-beta1 mRNA expression was up-regulated (AT: 208.5 +/- 79.3% vs. sham: 100 +/- 34.6% P< 0.05). Collagen and angiotensin II type 1 receptor (AT1R) expression were not significantly altered by AT. HEK-293 cells were used to determine the potential humoral factors involved in down-regulation of NEP. Application of aldosterone, ANP, asymmetric dimethylarginine, and angiotensin peptides failed to cause down-regulation of renal NEP expression in vitro. AT reduces NEP expression and stimulates TGF-beta1 signalling in the kidneys. Thus, even brief episodes of AT affect renal gene expression, which may account for structural renal changes and alterations of renal function in the long term.

METHYLATION STATUS OF NEUTRAL ENDOPEPTIDASE GENES DOWN‐REGULATED BY DIESEL EXHAUST PARTICULATES IN HUMAN AIRWAY EPITHELIUM

Our previous study has reported that epithelial neutral endopeptidase (NEP, a 90–110 kDa zinc‐dependent, type II integral membrane protein) expression was down‐regulated by diesel exhaust particulates (DEP) in a concentration‐dependent manner. In addition, sequence analysisof the NEP promoter reveals a CpG island in the type II promoterregion containing five Sp1 elements and juxtaposed to a region containing multiple Alu repeats. To determine the possibility thathypermethylation of the NEP promoter involved a NEP downregulation following exposure to DEP, we conducted a series of experiments in BEAS‐2B cells, including concentration‐effect and time‐course design (7 days). Methylation‐specific PCR primers were designed to discriminate between methylated and unmethylated alleles after bisulfite treatment. Our data show that there is no evidence of methylation in DNA samples either from the dose‐effect (0, 5, 10, 20, 40, 60 μg/ml DEP for 24 hours or from the time‐course designs (2, 4, and 7 days at the concentrations of 40 μg/ml DEP), suggesting that methylationof the tested NEP 5′ CpG island region could not be associated with loss of NEP expressionin epithelial cells after DEP exposure. However, a more comprehensive analysisof epithelial specimens after long‐term DEP exposure is needed to fully rule out the effect of NEP promoter methylation (Supported by HEI).

Upregulation of intermedin and reduced neutral endopeptidase expression in hypertensive cardiac hypertrophy

Intermedin (IMD), a recently discovered vasodilator peptide, has potential to protect against ischemia–reperfusion injury and to attenuate ventricular remodeling in response to pressure overload. Its actions are mediated by the calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1–3). Using the spontaneously hypertensive rat (SHR) model at 20 weeks of age and the normotensive WKY control, the aim was to (i) verify oxidative stress and concentric hypertrophy and (ii) examine expression of IMD and receptor components and of neutral endopeptidase (NEP), probably an important mediator of IMD degradation.

The expression of Bacillus intermedius glutamyl endopeptidase gene in Bacillus subtilis recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

Overexpression of kidney neutral endopeptidase (EC 3.4.24.11) and renal function in experimental cirrhosis

Neutral endopeptidase degrades atrial natriuretic peptide (ANP) and bradykinin and may generate endothelin-1 from big-endothelin. In advanced cirrhosis, sodium retention is accompanied by elevated plasma ANP levels, and infusion of ANP causes hypotension, but in normal humans increasing the concentration of ANP through the inhibition of neutral endopeptidase, localized in renal proximal tubule cells, causes natriuresis without any arterial pressure drop. The purpose of this study was the assessment of kidney neutral endopeptidase expression and responses to candoxatrilat (a specific inhibitor of this enzyme) in rats with CCl4-induced cirrhosis. Two groups of control rats (n = 5) were injected with vehicle or 3 mg/kg candoxatrilat. Three groups of cirrhotic rats with ascites (n = 10) received vehicle alone or 3 or 10 mg/kg candoxatrilat. In cirrhotic rats, Western blot analysis revealed a 170% increase in renal neutral endopeptidase protein content (P < 0.03), mainly in the proximal nephron and macula densa, and both candoxatrilat dosages increased plasma ANP levels, urinary volume, and urinary excretion of sodium, ANP, and cGMP compared with vehicle alone (all P < 0.03). Candoxatrilat (10 mg/kg) also reduced tubular solute-free water reabsorption (P < 0.03) in cirrhotic rats, but renal blood flow, arterial pressure, and plasma renin activity were unaffected. Neutral endopeptidase inhibition has natriuretic and aquaretic actions in cirrhosis without any effect on blood pressure and kidney perfusion due to a significant overexpression of this enzyme in renal cortex.

Gene expression arrays reveal a rapid return to normal homeostasis in immunologically-challenged trophoblast-like JAR cells

The immunologic adaptations of pregnancy have come under increasing scrutiny in the past 15 years. Existing experimental evidence clearly demonstrates that placental trophoblasts play an important role in regulating immunologic/inflammatory responses at the maternal–fetal interface. We used a well-developed gene expression array to examine in greater detail the physiologic response of trophoblast-like choriocarcinoma cells to a model immunologic ‘challenge.’ We co-cultured PHA-activated or resting peripheral blood mononuclear cells (PBMC) with the human choriocarcinoma cell line JAR for time periods ranging from 2 to 18 h. Messenger RNA expression in the JAR cells was then assessed using a 21,329-gene microarray and novel biostatistical analyses that we have previously published. Patterns of differential gene expression were assessed using a commercial pathway analysis software program. Differences in gene expression between JAR cells cultured with activated PBMC (experimental samples) and JAR cells cultured with resting PBMC (control samples) were seen only at the 2 h time point. That is, multiple genes were transcribed in JAR cells in response to activated PBMC, but expression levels of the genes had all returned to baseline by 6 h. Molecular modeling demonstrated that the differentially expressed genes were largely associated with cell growth and differentiation. This model was confirmed by noting a two-fold increase in CD10/neutral endopeptidase expression (a marker for cell differentiation) in JAR cells incubated with media from activated PBMC compared with JAR cells incubated with resting PBMC. These findings support the hypothesis that there is a delicate immunologic milieu at the maternal–fetal interface that must be maintained. Immunologic/inflammatory challenge at the maternal–fetal interface is compensated by cellular mechanisms that work to reduce inflammation and rapidly restore immunologic balance.

Neutral Endopeptidase Activity is Increased in the Skin of Subjects with Diabetic Ulcers

Cutaneous sensory nerves mediate inflammation and wound healing by releasing neuropeptides, such as substance P, which stimulates pro-inflammatory responses by keratinocytes, fibroblasts, and endothelial cells. The cell surface enzyme, neutral endopeptidase, degrades substance P, thereby regulating its biologic actions. We hypothesized that neutral endopeptidase enzymatic activity is increased in chronic wounds and skin from subjects with diabetes. We compared cutaneous neutral endopeptidase expression and enzymatic activity between normal controls and diabetic subjects with neuropathy and chronic wounds. Skin samples from subjects with diabetes were taken at the time of amputation for nonhealing ulcers. Skin taken from the ulcer margin, 1 cm from the ulcer (adjacent), and from the most proximal region of the amputated leg were studied. Skin biopsies from the leg of healthy control subjects were also studied. Neutral endopeptidase was localized by immunohistochemistry in all tissue sections. Neutral endopeptidase activity was measured using a fluorimetric assay. The median neutral endopeptidase activity of the ulcer margin was 1.21 x higher (p>0.2) than adjacent skin, 5.26 (p<0.001) than proximal skin, and 15.22 x higher (p<0.001) than control skin. Adjacent skin had a median neutral endopeptidase activity 4.34 x higher (p<0.001) than proximal skin and 12.58 x higher (p<0.001) than control skin. The median neutral endopeptidase activity of proximal skin was 2.90 x higher (p<0.001) than control skin. This elevated neutral endopeptidase activity in the skin and chronic ulcers of subjects with diabetes combined with peripheral neuropathy may contribute to deficient neuroinflammatory signaling and may impair wound healing in subjects with diabetes.

Damage-Induced Neuronal Endopeptidase (DINE/ECEL) Expression Is Regulated by Leukemia Inhibitory Factor and Deprivation of Nerve Growth Factor in Rat Sensory Ganglia after Nerve Injury

Damage-induced neuronal endopeptidase (DINE) is a novel metallopeptidase and is expressed in response to various neuronal injuries. The expression regulation of DINE mRNA in the dorsal root ganglia (DRGs) after sciatic nerve injury is examined. A substantial increase of DINE mRNA expression was observed in relatively small-sized DRG neurons after nerve injury. The expression was observed in isolectin B4-negative and partly TrkA-positive neurons, and the expression profile was fairly similar to that of the neuropeptide galanin. More than 80% of DINE mRNA-positive neurons simultaneously demonstrated galanin immunoreactivity after nerve injury. In cultured DRG, DINE mRNA expression was enhanced by leukemia inhibitory factor (LIF) but not by other growth factors and cytokines. LIF treatment to rat sciatic nerve induced DINE mRNA expression in DRG without nerve injury, and, conversely, the intranerve injection of anti-gp130 antibody after sciatic nerve injury significantly inhibited the upregulation of DINE mRNA in DRG. Furthermore, nerve growth factor (NGF) deprivation, which can induce galanin expression, also enhanced DINE mRNA expression in vitro and in vivo. Both LIF application and NGF deprivation additively enhanced DINE expression in vitro. These results suggest that DINE gene expression is regulated separately by both LIF and NGF deprivation, and this regulation pattern is similar to that of galanin gene expression. Because both DINE and galanin have a neuroprotective function, their simultaneous induction may provide more successful protection for injured sensory neurons.

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells.

Jaspamide (asplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells.

Expression, Localization and Activity of Neutral Endopeptidase in Cultured Cells of Benign Prostatic Hyperplasia and Prostate Cancer

Neutral endopeptidase is a cell surface enzyme that cleaves and inactivates neuropeptides. Neutral endopeptidase is expressed by prostatic epithelial cells and is thought to have a key role in the growth of androgen independent prostate cancer. In contrast to the encouraging data dealing with neutral endopeptidase and prostate cancer progression, only few studies are available of the significance of neutral endopeptidase in prostatic stromal cells and benign prostatic hyperplasia (BPH). We report the expression and activity of neutral endopeptidase in human prostatic stromal cells. We recently established and characterized human prostatic stromal cells from BPH tissue. Reverse transcriptase-polymerase chain reaction, Western blot experiments and immunohistochemistry were performed to investigate the expression levels of neutral endopeptidase and show the cellular localization of the enzyme in these cells. Enzymatic neutral endopeptidase activity was evaluated using a colorimetric assay in nonstimulated cells, and after androgen and estrogen application. Stromal BPH cells showed strong expression of the neutral endopeptidase messenger RNA and protein, equaling the neutral endopeptidase expression level in prostate cancer cells. The neutral endopeptidase protein located in the plasma membrane of the cells showed a mean enzymatic activity plus or minus standard deviation of 75% +/- 2.5% compared with LNCaP prostate cancer cells. Interestingly enzymatic activity of the membrane bound neutral endopeptidase in stromal cells was not regulated by androgen and estrogen. These data suggest that neutral endopeptidase is expressed by prostatic stromal cells. In addition to its role in the progression of prostate cancer, it may also be involved in regulatory processes in the stroma, namely the degradation of neuropeptides released from prostatic neuroendocrine cells.

Upregulation of Neutral Endopeptidase Expression and Enzymatic Activity during the Differentiation of Human Choriocarcinoma Cells

Neutral endopeptidase (NEP)/CD10, a cell-surface peptidase degrading various bioactive peptides, is mainly present in syncytiotrophoblasts in the human placenta. However, the change in NEP expression upon trophoblast differentiation remains to be clarified. In the present study, we examined the expression of NEP in the differentiating trophoblast using the BeWo choriocarcinoma cell line as a model system. Under the normal culture conditions, NEP was very weakly expressed on most proliferating cytotrophoblastic BeWo cells, while a minority of the cell population (less than 5 per cent ), consisting of giant, multinucleated cells, clearly expressed NEP at the cell membrane. Treatment of BeWo cells with forskolin (FSK) for 48–72h resulted in an 11− to 44-fold increase in the level of hCG secretion and induced cell fusion leading to the formation of multinucleated syncytiotrophoblasts, indicating functional and morphological differentiation. Fluorescence-activated cell sorting (FACS) analysis revealed that treatment with FSK significantly increased the cell-surface protein expression of NEP on differentiating BeWo cells. Consistently, there was a significant increase in the NEP enzymatic activity after FSK treatment. The level of hCG secretion from the FSK-treated cells was further enhanced when the cells were treated in the presence of the NEP inhibitor phosphoramidon. Immunohistochemical analysis of normal chorionic villi and choriocarcinoma tissues revealed the localization of NEP in syncytiotrophoblastic cells, as opposed to weak or negative staining in cytotrophoblastic cells. These data demonstrate that induction of choriocarcinoma cell differentiation is associated with an increase of NEP/CD10 expression at the cell surface, suggesting a role of this enzyme in regulating differentiated trophoblast functions such as hCG secretion. NEP/CD10 may also be a new cellular differentiation marker of both the normal and neoplastic trophoblast.

Neutral Endopeptidase Expression and Distribution in Human Skin and Wounds

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

Neutral endopeptidase (NEP) expression in normal human skin and in selected disease states

Neutral endopeptidase (NEP) expression in normal human skin and in selected disease states

EGF and TGF-beta regulate neutral endopeptidase expression in renal vascular smooth muscle cells

We recently reported that neutral endopeptidase (NEP) expression on renal vascular smooth muscle cells (VSMC) was downregulated in the presence of serum. Here we examine the role of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta) in this downregulation and the consequences of the changes in NEP activity on their mitogenic effects. EGF inhibited NEP activity, whereas TGF-beta was stimulatory. Expression of the enzyme was studied by measuring the binding of [125I]RB-104, a specific NEP inhibitor, and the fluorescence intensity of NEP-labeled cells. Both parameters were decreased by EGF and were increased by TGF-beta. NEP mRNA expression in EGF-treated cells was reduced after 48 h. In contrast, it was increased in TGF-beta-treated cells. Interestingly, NEP inhibition influenced the mitogenic effect of EGF. Indeed, thiorphan, an NEP inhibitor, and an anti-NEP antibody decreased EGF-dependent [3H]thymidine incorporation and cell proliferation by approximately 50%. TGF-beta had no effect on VSMC growth. These results indicate that EGF but not TGF-beta participates in the downregulatory potency of serum on NEP expression in VSMC. They also demonstrate that the full effect of EGF on VSMC proliferation depends on intact NEP activity.

Evidence for a polyamine-mediated control of soluble nitrogen mobilization during post-clipping re-growth of white clover (Trifolium repens L.)

A putative contribution of polyamines to the control of peptidase activity expression during re-growth was studied in source organs (roots and stolons) of defoliated white clover (Trifolium repens L.). Endopeptidase activity increased in roots during the first 6 days following complete defoliation, while exopeptidase expression seemed to be restricted to the early hours of re-growth. These changes correlated with an immediate 80% decline in the content of total free polyamines, mainly represented by the diamine cadaverine. The inhibitory capacities of cadaverine and spermine were tested on enzyme activity in vitro in order to elucidate whether the endogenous polyamine level was associated with the cut-induced endopeptidase expression. Cadaverine seemed to inhibit endopeptidase activity of stolons but not root endopeptidase activity. These data support the view that polyamines may play a role in the regulation of peptidase expression in source organs of white clover during post-clipping re-growth. The existence of different endopeptidase isoforms in roots and stolons is discussed in relation to the molecular mechanisms by which polyamines may regulate their activities.