ARTICLE |
Correspondence to: Alfredo Martínez, Div. of Clinical Sciences, National Cancer Institute, NIH, 9610 Medical Center Dr., Rm. 300, Rockville, MD 20850-3300.
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Summary |
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Adrenomedullin (AM) is a multifunctional regulatory peptide that stimulates cyclic AMP production in many target tissues and is highly expressed in the lung. Analysis of the distribution of the recently cloned AM receptor (AM-R) by non-radioactive in situ hybridization revealed abundant expression in the basal cells of the airway epithelium and Type II pneumocytes. The expression of AM-R in the two cell types involved in epithelial regeneration of the lung suggests that AM may be relevant in such functions as organ development, wound repair, and epithelial turnover. AM-Rs are also synthesized in vivo and in vitro by a variety of tumor cells that also express the ligand, suggesting the existence of an autocrine loop that may be involved in tumor growth stimulation. The present findings suggest that the AM/AM-R regulatory system plays a major role in respiratory physiology and lung carcinogenesis and that new functions for AM remain to be identified. (J Histochem Cytochem 45:159-164, 1997)
Key Words: human lung, lung tumors, adrenomedullin receptor, growth regulation, in situ hybridization
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Introduction |
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Adrenomedullin (AM) is an -amidated 52-amino acid peptide originally detected in a human pheochromocytoma by its ability to activate platelet adenylate cyclase (
The biological effects of AM are mediated by elevating cellular cAMP levels through a G-protein-coupled mechanism (
Expression of the AM-R is particularly high in lung, in which Northern blot analysis reveals several mRNA species, of which the most prominent is a 1.8-KB transcript (
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Materials and Methods |
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Tissues
Ten normal lung specimens from patients who died without pulmonary involvement were provided by the Department of Pharmacology, University of Pittsburgh (protocol M1259). Eighteen additional formalin-fixed, paraffin-embedded blocks containing representative cases of lung tumors (Table 1) were obtained from the files of the NCI-Navy Medical Oncology Branch, National Naval Medical Center, Bethesda (protocol 83-15).
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Cell Culture
Twenty-six lung tumor cell lines were used in this study: NCI-H727, H679, H835, UMC11 (carcinoids), H23, H726, H2087, H1373, H2228, H920 (adenocarcinomas), H1404, H820, A549 (bronchioalveolar carcinomas), H520 (squamous carcinoma), H82, H446, N417, H187, H345, H209, H510, H889, H774, H123, H735, and N592 (small-cell lung carcinomas). Cells were grown in serum-free, hormone-free medium, as previously described (
RT-PCR
Three oligonucleotides were selected via DNASTAR software analysis of rat AM-R cDNA (Table 2) and were used as primers/probe for PCR and Southern blot experiments. An expected PCR product of 471 BP was indicative of AM-R message expression. Respective oligonucleotides were synthesized by Midland Certified Reagents (Midland, TX).
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Reverse transcription was performed using the SuperScript Preamplification System (Life Technologies; Gaithersburg, MD) per the manufacturers protocol. A Perkin-Elmer 9600 thermocycler was used in amplifying the gene products in the samples. All buffers, enzymes and nucleotides used were obtained from Applied Biosystems (Perkin-Elmer Cetus; Norwalk, CT). PCR products were analyzed electrophoretically using 1% agarose gels, and after ethidium bromide staining were visualized under UV light, followed by Southern analysis. Two PCR products (total human lung and cell line NCI-H720) obtained using the same primers have been previously cloned and sequenced, obtaining a high homology with the original rat clone (
Southern Blot Analysis
Gels were denatured in 1.5 M NaCl/0.6 M NaOH and 1.5 M NaCl/2 M Tris and DNA transferred overnight onto 0.2-µm nitrocellulose filters in 20 x SSC by standard capillary flow techniques. DNA was crosslinked to the filter at 80C under vacuum and incubated in hybridization buffer. The anti-sense nested probe was 32P-end-labeled by standard procedures. Hybridization with the probe was done overnight at 42C. Room temperature stringency washes were performed using 5 x SSC/0.1 % SDS and 1 x SSC/0.1 SDS. Filters were air-dried and 32P signal resolved by autoradiography at -80C, using Kodak XAR5 film.
In Situ Hybridization
Detection of the AM-R mRNA was performed using in situ hybridization as previously described (
Immunocytochemistry
In selected slides, immunocytochemistry for AM was performed after in situ hybridization for the receptor. As previously characterized rabbit anti-human AM antibody (
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Results |
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Normal Lung
In the upper airways, AM-R mRNA was localized in the cytoplasm of the basal cells of the epithelium (Figure 1A). Control slides, treated with the sense probe, showed some nonspecific staining in discrete cells of the connective tissue (Figure 1B) but clearly demonstrated the specificity of the staining with the anti-sense probe in the other structures. Surprisingly, the smooth muscle bundles of both the airway walls and the blood vessels did not show any hybridization signal. Immunocytochemical staining for AM after in situ hybridization showed the previously described pattern (
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Tumors
We studied 18 tumors of pulmonary origin with different diagnoses (Table 1). Of the 18 studied, 13 tumors (72%) were positive for AM-R by in situ hybridization. Some examples of these are shown in Figure 4 and Figure 5. Analysis of the production of AM-R mRNA in tumor cell lines by RT-PCR revealed that 15 of the 26 cell lines (58%) expressed detectable amounts of mRNA, as demonstrated by Southern blot (Figure 6).
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Discussion |
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We have shown, using in situ hybridization techniques, that AM-R is highly expressed in the lung, consistent with the results of the Northern blot data presented in the original description of the AM-R (
We have previously described the distribution of AM in the normal and malignant human lung. AM is produced by the airway epithelium, bronchial glands, neurons, endothelial cells, chondrocytes, macrophages, and smooth muscle cells, and is present in many tumors (-amidating mono-oxygenase, the enzyme complex responsible for amidation of regulatory peptides (
The expression of AM-R in basal cells and Type II pneumocytes, two cell types involved in regeneration of the epithelial layers of the lung (
The specific distribution of AM and its receptor in the airway epithelium suggests a new hormonal mechanism of action that might be important in the rapid response to tissue injury. AM is stored in the apical end of the ciliated cells and is secreted into the lumen of the organ, where it acts as an antimicrobial agent (Tom Walsh, Pediatric Oncology Branch, NCI, personal communication). Ligand-specific receptors, on the other hand, are located in the basal region of the epithelium and are separated from this source of AM by the intact epithelial layer. If an epithelial injury occurs (infection, chemical or mechanical damage), cell boundaries are disrupted and the luminal pool of AM becomes available for receptor binding. Given the mitogenic potential of AM, this results in a rapid proliferative response which eventually heals the wound, restoring epithelial integrity and re-separating the ligand from the receptor. The term "traumakines" would be appropriate for peptides displaying such behavior, with AM as the first identified member of this family. During normal growth conditions (epithelial turnover), the receptors could receive the ligand from mesenchymal components, such as smooth muscle cells, or from the interstitial fluid derived from the plasma, a recognized source of AM (
The co-expression of both the ligand and the receptor in tumors suggests that AM and its receptor are implicated in an autocrine mechanism, that may regulate cell growth (
An unexpected finding was the absence of a hybridization signal in the smooth muscle cells of the blood vessels and the bronchial walls (Figure 1A and Figure 1C). Smooth muscle cells are believed to express AM-R that mediate the vasodilatatory and bronchorelaxant responses to AM. There are several reports (e.g.,
In conclusion, our data clearly demonstrate the expression of AM/AM-R in normal and malignant lung. The regional distribution of these molecules, along with previously reported data on their trophic action, implicates this ligand/receptor relationship as a possible growth regulatory mechanism involved with normal epithelial turnover, wound repair, and carcinogenesis.
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Acknowledgments |
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We thank Dr Jill M. Siegfried (Department of Pharmacology, University of Pittsburgh) for providing the normal tissue, and Dr R. Ilona Linnoila (NCI) for help with the tumors.
Received for publication May 29, 1996; accepted October 14, 1996.
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