PERSPECTIVE
Neuroendocrine Differentiation, Neuropeptides, and Neprilysin

Andrea J. Cohen and York E. Miller

Department of Medicine, Denver Veterans Affairs Medical Center, Division of Pulmonary Sciences and Critical Care Medicine, Specialized Program of Research Excellence in Lung Cancer, University of Colorado Health Sciences Center, Denver, Colorado

Pulmonary neuroendocrine cells exhibit highly specialized differentiation with a neurosecretory phenotype and significant bioactive peptide hormone content. In addition to these functions, pulmonary neuroendocrine cells have been demonstrated to act as chemoreceptors for hypoxia (1). Specific pulmonary disorders, including bronchopulmonary dysplasia, bronchiectasis, cystic fibrosis, pulmonary hypertension, eosinophilic granuloma, respiratory bronchiolitis, emphysema, and chronic bronchitis have been associated with pulmonary neuroendocrine cell hyperplasia (reviewed in 2). A series of elegant studies (reviewed in 3) demonstrate a role for bombesin-like peptides in promoting fetal lung development and surfactant maturation. Recently, administration of antibombesin antibody in a baboon model of bronchopulmonary dysplasia has resulted in clinical and pathologic improvement (3). Thus, in the prenatal setting, neuroendocrine cell-derived bombesin-like peptides have a beneficial effect, whereas in the setting of bronchopulmonary dysplasia, excessive concentrations of the same peptides contribute to disease. Therefore, the control of pulmonary neuroendocrine cell numbers is clearly of clinical relevance. The processes of neuroendocrine cell differentiation, proliferation, and apoptosis combine to determine pulmonary neuroendocrine cell numbers. In this issue, Willett and colleagues (4) demonstrate that inhibition of neuropeptide degradation may increase neuroendocrine cell hyperplasia, perhaps by an autocrine effect.

The stem cells that act as progenitors for airway neuroendocrine cells are not fully defined, nor are the cells into which the airway neuroendocrine cell may transdifferentiate. The recent report of dual CC10/CGRP positive cells in the mouse airway after injury suggests a close relationship between the Clara and neuroendocrine cell lineages (5). Some of the transcriptional events regulating pulmonary neuroendocrine cell differentiation are beginning to be defined. Members of the achaete-scute family of basic helix-loop-helix transcription factors play critical roles in the development of neuronal structures in Drosophila as well as vertebrates. Mice homozygous for null mutations in the mouse achaete-scute homologue-1 (MASH1) exhibit multiple abnormalities in olfactory and enteric neurons and die within 24 h of birth. Of interest, MASH1 ^/ mice have no pulmonary neuroendocrine cells but do have gastrointestinal neuroendocrine cells, demonstrating a necessary and surprisingly specific role for MASH1 in pulmonary neuroendocrine cell differentiation (6). The human achaete-scute homologue-1 (hASH1) is expressed in small-cell lung carcinomas (SCLC) with neuroendocrine differentiation, but not in non-small-cell lung carcinomas (NSCLC). Depletion of hASH1 messenger RNA (mRNA) with antisense oligonucleotides results in a loss of neuroendocrine features (6). In Drosophila, neural fate is specified by inhibition of achaete-scute expression through the action of hairy and other enhancer-of-split transcriptional repressors. Expression of a human hairy homologue, hairy-enhancer-of-split-1 (HES-1), correlates negatively with hASH1 expression and neuroendocrine phenotype in lung cancer cell lines (7). Enforced expression of HES-1 in a SCLC cell line represses hASH1 expression.

Careful morphometric studies in animal models have demonstrated that pulmonary neuroendocrine cell hyperplasia caused by airway epithelial injury is accompanied by a combination of increased proliferation (4, 8) and decreased apoptosis (4) of neuroendocrine cells. One interesting model of cell cycle and apoptosis deregulation is the retinoblastoma (Rb)^+//p53^/ mouse, which exhibits lymphomas, pituitary, thyroid, and islet cell tumors (9). In addition, these mice have a high incidence of pulmonary neuroendocrine cell hyperplasia, which has been compared to idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells in humans (10). Analysis of the hyperplastic pulmonary neuroendocrine cell aggregates in the Rb^+//p53^/ mouse reveals a further somatic mutation with loss of the remaining active Rb allele, similar to genetic defects commonly seen in human SCLC (Carl Hilliker, Harry Drabkin, and Robert Gemmill, personal communication). The multiple endocrine neoplasia type 1 syndrome is frequently accompanied by bronchial carcinoids. Thus, the MEN1 tumor suppressor gene is potentially more specifically related to pulmonary neuroendocrine cell growth regulation than either p53 or Rb. MEN1 has no obvious homologies to suggest function but has recently been demonstrated to interact with JunD and to repress JunD-activated transcription (11).

Neuropeptides may be important in regulating pulmonary neuroendocrine proliferation and survival. This idea is an extrapolation from the neuropeptide autocrine growth regulation seen in SCLC. Bombesin-like peptides were the first autocrine growth factors demonstrated to act in a human malignancy (12). Attempts to interrupt neuropeptide autocrine growth regulation in SCLC in a clinical setting have been hampered by the redundancy of different neuropeptides involved. Strategies to overcome this redundancy have achieved some success. Substance P analogues, which interact with multiple neuropeptide receptors, have been synthesized. Rather than acting as competitive blockers, the substance P derivatives are biased agonists, inhibiting some postreceptor pathways (Ca²⁺ mobilization, ERK family kinases) and activating others (JNK family kinases). The net effect is to initiate apoptosis (13). Another means of manipulating the action of multiple neuropeptides is to alter the expression of neprilysin (neutral endopeptidase [NEP], CD10, E.C.3.4.24.11), a cell surface peptidase, which hydrolyzes small peptides at the amino side of hydrophobic residues. The range of substrates cleaved by NEP is broad and includes bombesin-like peptides, bradykinin, substance P, atrial naturetic peptide, enkephalins, endothelin, and the oxidized  chain of insulin. NEP is expressed at very low or undetectable levels in both SCLC and NSCLC (14, 15). The mechanism of inactivation is not mutational (15). Inhibition of NEP potentiates neuropeptide-induced Ca²⁺ mobilization. Exogenous administration of NEP can inhibit lung cancer growth, either in vitro or in immunodeficient rodent tumor xenograft models (16). Thus, NEP is a negative growth regulator for many lung cancers, although in the absence of mutations it is not a classical tumor suppressor.

Willett and colleagues have demonstrated parallel increases in NEP expression and neuroendocrine cell numbers in an animal model of neuroendocrine cell hyperplasia (4). They have also shown that NEP inhibition leads to a further increase in neuroendocrine cell hyperplasia and proliferation, and a decrease in apoptosis. NEP has multiple substrates, so potentiation of autocrine growth factors remains an unproven but appealing hypothesis for this effect. In several human lung diseases characterized by pulmonary neuroendocrine cell hyperplasia, including idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells (10, 17), respiratory bronchiolitis and eosinophilic granuloma (Andrea Cohen and York Miller, submitted manuscript), NEP expression is also increased and may act in a counterregulatory fashion to mitigate high levels of neuropeptides. In summary, NEP expression is decreased in many malignancies driven by autocrine neuropeptides but appears to be frequently increased in benign conditions exhibiting neuroendocrine cell hyperplasia. These differences may represent disrupted or intact feedback mechanisms in malignant and benign disorders, respectively.

NEP expression is modulated by various factors, including interferon, interleukin (IL)-1, tumor necrosis factor (TNF), IL-4 and glucocorticoids (18). Tobacco smoke has been reported to inactivate NEP in vitro and in an animal model, but studies in humans have not demonstrated a clear difference in NEP activity between smokers and nonsmokers (reviewed in 19). Genetic variation in NEP could account for some of the wide variation observed in humans (15). Mice homozygous for NEP null mutations are surprisingly normal but do exhibit decreased blood pressure, increased microvascular permeability, and increased susceptibility to endotoxin shock (20). The hypothesis that genetic variation in NEP expression may cause differences in susceptibility to disorders such as bronchopulmonary dysplasia, lung cancer, asthma, and sepsis, in which neuropeptides have been implicated, remains untested but attractive.

	Footnotes

Address correspondence to: York E. Miller, M.D., RESP 111A, Denver Veterans Affairs Medical Center, 1055 Clermont St., Denver, CO 80220. E-mail: york.miller{at}uchsc.edu

(Received in original form May 6, 1999).

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