Published ahead of print on November 1, 2007, doi:10.1165/rcmb.2007-0388TR
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0388TR
Pulmonary Genomics, Proteomics, and PLUNCs1 Academic Unit of Respiratory Medicine, University of Sheffield Medical School, and 2 Department of Oral Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, United Kingdom Correspondence and requests for reprints should be addressed to Colin D. Bingle, Ph.D., Academic Unit of Respiratory Medicine, Section of Infection, Inflammation and Immunity, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2JF, UK. E-mail: c.d.bingle{at}sheffield.ac.uk
Key Words: genomics • proteomics • pulmonary • plunc Over the past decade Pulmonary Medicine, like many biomedical research fields, has benefited enormously from the major advances that have occurred in genomic and proteomic techniques. The relative abundance of potential samples including lung tissue and cells, bronchoalveolar lavage fluid (BALF), and sputum make pulmonary conditions attractive for many profiling studies, which are ultimately aimed at identifying novel diagnostic and prognostic disease markers. Thankfully, these studies have largely confirmed the presence of many expected proteins as well as the identification of novel genes and proteins. These studies do, however, lend themselves to the development (in researchers) of one of two conditions named and identified by Naftali Kaminski as AGLSS (Acute Gene List Staring Syndrome) and CGLSS (Chronic Gene List Staring Syndrome). Both conditions are characterized by sleep deprivation, refusal to return to the bench, and excitement about funny gene names, but the chronic condition may also involve paralysis of career (what is now called Kaminski's tetrad). Notwithstanding the above concerns, it is beyond doubt that the use of unbiased proteomic and genomic analyses have led to the identification of many novel molecules which by the very means of their identification are likely to play roles in the biology of the respiratory tract. In these studies there is often a selection of proteins that are consistently observed to be present and/or differentially expressed. In this short overview we will focus on members of a novel family of proteins, the PLUNCs, which may be considered as a paradigm for such gene/protein identification.
Palate, lung, and nasal epithelium clone (plunc), was initially isolated by differential display in a study designed to identify molecules associated with palate closure in the mouse (1). It transpires, however, that the gene is not expressed in the palate per se but rather is found in the developing nasal passages and, perhaps of greater interest to pulmonary biologists, it is highly expressed in the tracheal epithelium and main bronchi, with expression rapidly lost with distal bronchial branching (1). This distribution pattern appears to be unique among the major characterized proteins of the respiratory tract. Within a short space of time we (2) and others (3–5) reported the cloning of human PLUNC. Unfortunately, each group gave the gene a different name: YY1 (3), Lunx (4), Spurt (5). These initial studies appeared to confirm that the human gene also had a similarly restricted expression pattern to that originally described for the mouse. Subsequently, we identified a family of PLUNC-related sequences and were able to define the human PLUNC locus on chromosome 20q11.21 that has evolved through a series of gene duplication events (6, 7). Analysis of these proteins has shown that their predicted structures are similar to that of lipopolysacharride (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), two essentially antagonistic, mammalian proteins critical in the mediation of signals from LPS. This similarity has led us to speculate that the PLUNC family also have a host defense function. PLUNCs, which compose the largest branch of the BPI/LBP family, can be divided into two subgroups, the short (S) and long (L) PLUNCs. SPLUNCs have homology to the N-terminal domain of BPI/LBP, while LPLUNCs have homology to both domains (6–8). Using this nomenclature, PLUNC (YY1, Lunx, spurt) has become SPLUNC1 (6, 7). Currently there is evidence for nine human PLUNCs (four SPLUNCs and five LPLUNCs) as well as at least one LPLUNC pseudogene (LPLUNC5). Molecular phylogenetic analysis has shown that PLUNCs are among the most rapidly evolving mammalian genes (7), as befits proteins performing a host defense function. We have also characterized PLUNC loci in mice, rats, and cows (7, 9), and this analysis has shown that these genes also exhibit a high level of interspecies diversification, with the greatest differences being found in the most recently evolved SPLUNC portion of the gene family. The biological function of PLUNCs remain unresolved but is now gaining the attention of a number of researchers, largely through their identification in many genomic and proteomic studies. Of all the different PLUNCs in both humans and rodents, it is SPLUNC1 and LPLUNC1 that are identified most frequently. Perhaps one of the most striking of these studies is that of Ross and coworkers (10) describing the transcriptional changes exhibited by tracheobronchial epithelial (TBE) cells as they undergo mucocilliary differentiation, which showed that LPLUNC1 (identified only as an uncharacterized affymatrix probe) and SPLUNC1 were the two most differentially expressed genes.
As previously outlined, mouse plunc was initially identified by RNA differential display from embryonic mouse palatal shelves and nasopharyngeal epithelium (1). The human gene was subsequently identified using the same technique (4, 5), as well as by suppression subtraction PCR (11), direct expressed sequence tag (EST) sequencing from nasopharyngeal cDNA libraries (3, 12), and by EST analysis (2). Similar techniques have also identified this gene in rats, cows, and pigs (13–15). At the molecular level splunc1 has been shown to be down-regulated in a number of mouse studies, for example in a range of experimental asthma studies (16, 17) as well as in a model of obliterative airway disease (18). In contrast, it is significantly induced in the respiratory epithelium of the nasal cavity following olfactory bulbectomy in rats (13). At the protein level, SPLUNC has been identified in nasal lavage (19), nasal mucous (20), BAL (21), and sputum (5, 22), and is one of the major secretory products of TBE cells grown at the air–liquid interface (ALI) (23). Analysis of nasal cells from patients with and without cystic fibrosis (CF) has shown elevated levels of SPLUNC1 in CF (24). This is consistent with other studies showing elevated levels of SPLUNC1 in chronic obstructive pulmonary disease and emphysema (5). Lung irritants also cause an up-regulation of SPLUNC1, as seen in nasal samples from smokers and epoxy workers (19, 25), and levels are also altered in allergic rhinitis (26). These results support the suggestion that SPLUNC1 is involved in inflammatory processes in the upper airways. The cellular source of SPLUNC1 within the respiratory tract has been shown to be a population of nonciliated airway cells (2, 5, 27) as well as the submucosal glands of the human upper airways (5, 23). More recently we and others have shown that in addition to these locations, the minor glands of the nasal epithelium (27, 28) and the oral cavity are also major sources of SPLUNC1 (27). We also demonstrated SPLUNC1 expression in lung carcinomas with a glandular phenotype highlighting SPLUNC1 as a possible lung cancer marker (27). This observation is supported by the identification of SPLUNC1 (LUNX) as a putative marker for non–small cell lung cancer (4, 29), and as a possible marker head and neck squamous cell carcinoma (30).
Fewer studies have been performed specifically on LPLUNC1, yet it is also frequently identified as a major gene product in pulmonary array studies. We initially identified LPLUNC1 from EST and bioinfomatic analysis and cloned the cDNA from tracheal tissue. Before this, the mouse ortholog was deposited in Genbank (accession number U46068) as a gene product expressed in the von Ebner's glands of the dorsal tongue and was christened von Ebner minor salivary gland protein. The first reported observation of this transcript was in a Representational Difference Analysis (RDA) study designed to identify human genes differentially expressed between fetal and adult lung (31). Since this time, LPLUNC1 has also been identified as a highly expressed transcript in EST studies of genes present in the nasopharyngeal epithelium (11, 12) and in airway epithelial cells isolated from patients with CF (32). Consistent with mouse in situ hybridization data (33), recent serial analysis of gene expression studies have confirmed that LPLUNC1 is predominantly expressed in the bronchial epithelium compared with peripheral lung tissue (34). In agreement with the markedly increased expression shown by Ross and colleagues (10) as TBE cells undergo mucocillary differentation, lplunc1 is also elevated in multiple studies of models of allergic asthma, suggesting an association with the asthmatic phenotype (16, 17). There are no published studies of LPLUNC1 protein localization. However, as with SPLUNC1, there are many reports of the protein in proteomic studies. For example LPLUNC1 has been identified in nasal mucous (35), BAL (21), and sputum (22). In BAL, LPLUNC1 levels were increased in patients with asthma after segmental allergen challenge (21). Recently LPLUNC1 was also shown to be a major secreted product of TBE cells cultured at the ALI and was decreased by IL-4 treatment (36).
It is increasingly evident that genomic and proteomic profiling techniques are powerful tools for the identification of novel molecules and the validation of these molecules as tools to study the molecular changes associated with pulmonary disease. Although these experimental techniques have their weaknesses with regard to their sensitivity and reproducibility, when specific molecules are identified across a wide range of biological samples using platforms that study both gene and protein products, then perhaps one can have increased confidence that these molecules are worthy of additional study. In this overview we have shown how members of the newly described family of putative defense molecules, PLUNCs are commonly identified in such studies using multiple biological samples from the respiratory tract. Despite having as yet undefined functions, we suggest that this family will increasingly attract the interest of the wider pulmonary community.
Originally Published in Press as DOI: 10.1165/rcmb.2007-0388TR on November 1, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form October 29, 2007
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THE PLUNC FAMILY
SPLUNC1 (PLUNC)
