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Immunohistochemical Distribution of Sphingosine Kinase 1 in Normal and Tumor Lung Tissue

Korey R. Johnson, Kristy Y. Johnson, Heather G. Crellin, Besim Ogretmen, Alice M. Boylan, Russell A. Harley and Lina M. Obeid

Division of General Internal Medicine, Ralph H. Johnson Veterans Administration Hospital, Charleston, South Carolina (AMB,LMO); Department of Medicine (KRJ,HGC,LMO), Biochemistry and Molecular Biology (BO,LMO), Department of Medicine (AMB), Division of Pulmonary Medicine and Critical Care, and Department of Pathology and Laboratory Medicine (RAH), Medical University of South Carolina, Charleston, South Carolina; and the Department of Biology, The Citadel, Charleston, South Carolina (KYJ)

Correspondence to: Lina M. Obeid, MD, Department of Medicine, Medical University of South Carolina, 114 Doughty St., PO Box 250779, Charleston, SC 29425. E-mail: obeidl{at}musc.edu

	Summary

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Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type–specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient matched non–small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [10 samples], and adenocarcinoma tumors [10 samples]), exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer. (J Histochem Cytochem 53:1159–1166, 2005)

Key Words: sphingosine kinase 1 • immunolocalization • lung • non–small-cell lung cancer

	Introduction

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SPHINGOLIPIDS, ORIGINALLY THOUGHT OF as strictly structural components of the plasma membrane, have recently emerged as important bioactive lipids that influence cellular processes such as cell growth and survival, as well as programmed cell death. Central to the bioactive sphingolipid metabolic pathway are the metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P). Although ceramide and sphingosine have been shown to arrest cell growth and promote apoptosis, S1P has been demonstrated to stimulate cell growth and prevent apoptosis (Futerman and Hannun 2004).

S1P is formed by the enzyme sphingosine kinase (SK) catalyzing the phosphorylation of sphingosine. There are two main isoforms of SK, termed SK1 and SK2. Where SK1 has been associated with promoting cell growth, stimulating tumorigenesis, and facilitating angiogenesis (Olivera et al. 1999; Xia et al. 2000; Ancellin et al. 2002), SK2 has been implicated in arresting cell growth and promoting apoptosis (Igarashi et al. 2003; Liu et al. 2003). The enzymes also differ in their tissue distribution, with SK1 mRNA expression at its highest in the lung, spleen, and peripheral blood leukocytes (Melendez et al. 2000), whereas SK2 mRNA is expressed highest in the liver, brain, and heart (Liu et al. 2000). Intriguingly, Billich and colleagues found in homogenized mouse tissue SK1 activity was between 3–20 times higher than SK2 in the majority of tissues screened (Billich et al. 2003). These studies revealed the highest SK1 activity to be in lung, spleen, and the blood, whereas SK2 activity was highest in the blood, with intermediate levels of activity found in the spleen, brain, liver, lung, and lymph node.

Recent immunohistochemical studies by Murate and colleagues, using a rabbit polyclonal antibody directed against the C terminus of mouse SK1 and shown to cross-react with human SK1, found strong positive staining in such tissues as the cerebrum, cerebellum, midbrain, kidney, endothelial cells of blood vessels, megakaryocytes, and platelets (Murate et al. 2001). In addition, moderate to weak staining for SK1 was found in liver, spleen, intestine, and testis. In this study, an immunospecific antibody raised against the last 20 amino acids of the C terminus of human SK1 was used to examine lung tissue in detail for cell type–specific SK1 expression by immunohistochemical methods. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient-matched lung tissue and found in carcinoid (5 samples), squamous (10 samples), and adenocarcinoma tumors (10 samples); 25 of 25 tumor samples screened exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer.

	Materials and Methods

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Preparation of Rabbit Polyclonal Antibody Against SK
The antibody was prepared by the Medical University of South Carolina antibody facility. Briefly, a synthetic oligopeptide corresponding to the last 20 amino acids of the C-terminal (CVEPPPSWKPQQMPPPEEPL) of the hSK1 (GenBank accession no. AAF73423) was conjugated to keyhole limpet hemocyanin and injected into New Zealand White rabbits. Antiserum was affinity purified over a cyanogen bromide–activated agarose column bound with the same oligopeptide and eluted with 100 mM glycine (pH 2.5). Immunospecificity of the antibody for human SK1 was tested by immunoblot analysis and confirmed by immunoabsorption using the synthetic oligopeptide against which the antibody was raised (Johnson et al. 2002).

Immunohistochemistry
Paraffin blocks of normal and tumor tissue from lung cancer patients were obtained from the Medical University of South Carolina/Hollings Cancer Center Tumor Bank. After sectioning onto microscope slides, tissue sections were deparaffinized in xylene and rehydrated in a series of ethanol dilutions. Sections were incubated 10 min in 3% hydrogen peroxide to quench endogenous peroxidase. To improve antigen retrieval, sections were incubated in Vector Antigen Unmasking Solution (Vector Laboratories Inc.; Burlingame, CA) for 30 min in a warm humidifier chamber. After washing in PBS, sections were blocked 30 min in 2% normal goat serum in PBS. Sections were then incubated for 1 hr at room temperature with primary rabbit anti-hSK1 antibody (7 µg/ml in PBS) alone or combined with 1 µg/ml of immunizing synthetic peptide (incubated 1 hr before adding to the sections), followed by 30 min incubation with diluted biotinylated secondary antibody and then 30 min incubation with VECTASTAIN ABC Reagent (Vector Laboratories Inc.). Diaminobenzidine/H₂O₂ was used as a substrate for the immunoperoxidase reaction. Sections were lightly counterstained with hematoxylin, rehydrated, and mounted for analysis by bright field microscopy. In the case of immunoabsorbed antibody incubations, 1 µg of synthetic oligopeptide against which the antibody was raised against was incubated with 1 ml of diluted rabbit anti-SK1 antibody (7 µg/ml in PBS) for 1 hr at room temperature.

Dot-blot Analysis of SK1 mRNA Levels
Analysis of SK1 mRNA levels in various solid tumors were examined using cDNA Cancer Profiling Array (Clontech; Mountain View, CA) containing normalized cDNA collected from tumor and corresponding normal adjacent tissue from 241 patients. A 1.1-kb fragment corresponding to human SK1 open reading frame was used to generate a cDNA probe by [³²P]dCTP random priming. The profiling array was hybridized overnight in ExpressHyb buffer (Clontech) containing the ³²P-labeled SK1 cDNA probe at 68C and washed according to manufacturer's protocol. The array was exposed to X-ray film and the spots were quantified by densitometry. The data were normalized for each individual tumor based on the expression of SK1 in matched normal adjacent tissue.

	Results

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Immunohistochemical Staining of Normal Adjacent Lung Tissue
The distribution of SK1 was examined in several formalin-fixed paraffin-embedded tissue sections from tumor-free regions of the lung. The sections of the lung contain branching bronchioles that ultimately lead to terminal or respiratory bronchioles with adjacent alveolar walls and small to medium pulmonary arteries and veins.

In Figure 1A, SK1 staining was seen in the bronchiole. Normal pseudostratified columnar epithelial cells stained very intensely on the apical surface at the point of ciliary attachment continually throughout the basal plate of the bronchiole (Figure 1A, solid arrow). These epithelial cells also consistently demonstrated diffuse cytosolic staining throughout the bronchiole. Furthermore, punctate cytosolic staining was evident in the smooth muscle cells subjacent to the epithelium (Figure 1A, open arrow).

View larger version (144K): [in this window] [in a new window]   Figure 1 Immunohistochemical detection of sphingosine kinase 1 in histologically normal human lung tissue. Paraffin-embedded tissue sections were immunostained as described in Materials and Methods. Positive immunostaining was evident in (A) pseudostratified columnar epithelial cells (solid arrow indicates concentrated staining at the basal plate of the bronchiole) and smooth muscle cells (open arrow), (B) serous glands (solid arrow), (C) immature chondrocytes (solid arrow), (D) type II alveolar cells (solid arrow) and foamy macrophages (open arrow), (E) endothelial cells (closed arrow) and smooth muscle cells (open arrow) in arterioles and venules, and (F) nerve bundles (solid arrow). Bars = 20 µm.

Figure 1C shows staining of hSK1 in bronchiolar cartilage. Interestingly, SK1 levels appear to be related to chondrocyte maturation. Although immature chondrocytes showed moderate staining for SK1, mature chondrocytes were devoid of staining.

Continuing along the respiratory tract, in Figure 1D, SK1 was stained in terminal alveoli. Moderate cytosolic staining was evident in type I alveolar cells, whereas intense cytosolic staining was evident in type II alveolar cells (Figure 1D, solid arrow). Furthermore, intense staining was also seen in foamy macrophages present in the alveolar space (Figure 1D, open arrow).

Figure 1E shows the staining pattern for SK1 in bronchiolar blood vessels. Intense cytosolic staining of luminal endothelial cells was found in both the vein and nearby arterioles (Figure 1E, closed arrow). Additionally, smooth muscle cells surrounding both of these vessels showed moderate cytosolic staining for SK1 (Figure 1E, open arrow).

In Figure 1F, we stained for SK1 in bronchiolar nerve bundles. Surprisingly, intense cytosolic staining of nerve bundles was evident throughout the lung (Figure 1F, solid arrow).

Immunohistochemical Staining of Lung Cancer Tissue
We examined 5 carcinoids, 10 squamous cell carcinomas, and 10 adenocarcinomas for their expression of SK1 as compared with normal adjacent tissue. Overall, 25 of 25 tumor samples screened exhibited overwhelmingly positive immunostaining for SK1. Specificity of the rabbit polyclonal antibody for SK1 is seen in Figure 2A, in which light positive staining is evident in normal type I and II alveolar cells. In Figure 2B, an identical serial section of tissue probed with anti-SK1 antibody preabsorbed with the immunization oligopeptide is totally devoid of staining. Staining of a typical carcinoid tumor (Figure 2C) clearly illustrates intense immunopositive staining of the cancerous lesion with a defined border, whereas this staining was devoid in the surrounding stroma. Individual carcinoid tumor cells in this section displayed an evenly diffuse cytosolic staining pattern for SK1. Immunospecific detection of SK1 can be seen in Figure 2D, in which a serial section of the same carcinoid tumor probed with anti-SK1 preabsorbed with the immunization peptide lacked staining altogether. Staining in all of the squamous cell carcinomas examined revealed a strong positive cytosolic pattern uniform in the tumor cells throughout the lesion that was conspicuously absent in the surrounding stroma (Figure 2E). Collectively, all of the adenocarcinomas screened stained much stronger for SK1 than did the surrounding normal lung tissue. Staining patterns in individual adenocarcinoma cells differed somewhat from carcinoid and squamous cell types, in that there was light, diffuse cytosolic staining with quite distinct intense staining at the apical surface of the cells lining the lumen of the lung (Figure 2F).

View larger version (127K): [in this window] [in a new window]   Figure 2 Immunohistochemical detection of sphingosine kinase 1 (SK1) in non–small-cell lung carcinoma. Light immunopositive staining was evident in normal lung tissue (A), whereas cancerous lesions in the lung displayed strong immunopositive staining: (C) carcinoid, (E) squamous cell, and (F) adenocarcinoma (solid arrow indicates concentrated apical staining in these cells). (B,D) Blocking of staining in a normal and carcinoid tumor section with preabsorbed anti-SK1 antibody as described in Materials and Methods. Bars = 20 µm.

View larger version (27K): [in this window] [in a new window]   Figure 3 Sphingosine kinase 1 (SK1) mRNA is elevated in lung cancer and several other solid human tumors. (A) 32P randomly labeled SK1 cDNA probe was used to analyze SK1 mRNA levels in matched Tumor/Normal cDNA Profiling Array (Clontech). The data were normalized for each individual tumor based on the expression of SK1 in matched normal adjacent tissue. The number of patients analyzed for each tumor type is indicated in the graph (n) and data are expressed as the mean ± SD (p0.05). (B) In the same cDNA array, SK1 mRNA levels were examined for individual patients with lung cancer. Each bar corresponds to individual patient's fold change in relative SK1mRNA level between normal adjacent tissue and tumor tissue. Bold hatched line represents an average of 2-fold elevation of SK1 expression in tumor versus normal tissue. In all, 20 patients matched normal/tumor tissue were examined.

	Discussion

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One of the most interesting findings in this study was the intense staining for SK1 found in the basal plate of ciliated epithelial cells in the bronchiole (Figure 1A). The concentration of SK1 at the basal plate of ciliated epithelial cells may indicate a role for SK1 in ciliary movement to aid in the mucociliary clearance in the lung. In addition, the concentration of SK1 in the apical region of the pseudostratified epithelial cells may indicate a role of SK1, and ultimately S1P production, as an inflammatory mediator to aid in the pulmonary host defense mechanism. In fact, S1P was shown recently to stimulate interleukin 8 secretion in airway epithelial cells, thus suggesting a role for S1P-mediated signaling in airway inflammatory reaction (Cummings et al. 2002). In the submucosal layer, located directly beneath the mucosa layer of pseudostratified epithelial cells, punctate cytoplasmic staining was evident in smooth muscle cells. This is not surprising in light of several studies implicating S1P-mediated mitogenic signaling in cultured airway smooth muscle cells (Waters et al. 2003).

Adjacent to the bronchiole, prominent staining for SK1 was evident in the serous glands but absent in the mucous glands. Interestingly, we previously found that the cystic fibrosis transmembrane conductance regulator, also mainly localized to serous cells in airway mucosal glands, could regulate the transport of S1P across the plasma membrane of the cell (Boujaoude et al. 2001). Thus it is conceivable that the cystic fibrosis transmembrane conductance regulator may also regulate the release of S1P produced by the abundance of SK1 found in the cytoplasm of these serous cells. In addition, serous cells secrete a rich mixture of proteins that have antimicrobial, antiprotease, antioxidant, and anti-inflammatory functions. Because S1P has been implicated as a physiological activator of both alveolar macrophages and mast cells (Hornuss et al. 2001; Jolly et al. 2002), it is possible that the abundance of SK1 in serous cells produces S1P that is secreted in to the lung to aid in antimicrobial response, whereas the cystic fibrosis transmembrane conductance regulator aids in transporting S1P into the cells to control the inflammatory response that may be overstimulated with excess S1P in extracellular spaces within the lung.

Next to the bronchus, immature chondrocytes in the hyaline cartilage contained copious amount of positive staining for SK1 that appears to subside as the chondrocytes mature, because they are devoid of positive staining. Thus SK1-mediated synthesis of S1P may play an important part of the maturation process that is no longer needed as the chondrocyte reaches maturity. A recent report by Chae and colleagues describe aberrant chondrocyte condensation in the limbs of Edg1-knockout mice, which lack one of the transmembrane receptors for S1P, suggesting S1P mediated signaling is important to this developmental process (Chae et al. 2004).

As the respiratory bronchiole terminates into alveolar sacs, intense positive staining for SK1 was evident in type II alveolar cells. Because type II alveolar cells are known to secrete lung surfactant containing growth factors and cytokines that alter the inflammatory process, perhaps elevated SK1 expression and ultimately S1P production is part of the protein-lipid complex secreted by these type II alveolar cells to control the inflammatory response. Throughout the alveolar space, numerous alveolar macrophages stained positively for SK1, suggesting a role for SK1 in the activation of alveolar macrophages. In fact, it was recently shown that S1P in alveolar macrophages induced respiratory bursts, suggesting a physiological role for S1P as an activator of alveolar macrophages (Hornuss et al. 2001).

Prominent staining for SK1 was evident in endothelial cells and smooth muscle cells of venules and arterioles located throughout the lung. Recent reports show that S1P, the metabolic product of SK1, can enhance the pulmonary endothelial cell barrier, indicating an important role for S1P in the regulation of pulmonary vascular permeability (Dudek et al. 2004). In addition, S1P-mediated signaling has been shown to stimulate proliferation of vascular smooth muscle cells and constriction of the cells to aid in the regulation of vascular permeability (Lockman et al. 2004; McVerry and Garcia 2005).

Nerve bundles found throughout the lung contained a definite positive staining for SK1. SK1 expression and ultimate S1P production in these nerve bundles may serve as a cytoprotective effect to prevent apoptosis (Edsall et al. 1997), or quite possibly S1P production may play a role in neurogenesis (Harada et al. 2004).

One of the most intriguing findings in our immunohistochemical staining for SK1 in lung tissue was the overwhelmingly positive staining for SK1 in all of the carcinoid, squamous, and adenocarcinoma cancerous lesions, as compared with nearby adjacent tissue. Screening of a cDNA cancer-profiling array confirmed that SK1 mRNA is also elevated approximately 2-fold in patients with squamous and adenocarcinoma lung tumors, as compared with patient-matched normal adjacent lung tissue (Figure 3). Recent studies by Xia and colleagues show SK1 can directly transform cells and suggest an oncogenic role for SK1. Thus lung carcinomas may selectively upregulate SK1 to promote tumorigenesis (Xia et al. 2000).

In summary, we found very distinct positive staining for SK1 in a variety of normal cell types in the lung. In general, the normal cells that stained immunopositive for SK1 were cell types that play a role in host defense against microbial insult and secrete factors that regulate the inflammatory response. In addition, we observed a distinct strong immunopositive staining for SK1 in the cancerous lesion in a variety of non–small-cell carcinomas of the lung as compared with normal adjacent tissue, and this trend in elevated SK1 expression in lung cancer tissue was confirmed at the mRNA level. Taken together, these findings provide clues as to how SK1 may play a vital role in normal lung physiology and may ultimately be exploited in cancerous lesions to stimulate aberrant tumor growth.

	Acknowledgments

 
This work is supported by National Institutes of Health Grant GM-62887 and PO1 CA-097132 (to LMO).

We thank Dr. Debra J. Hazen-Martin, Ms. Margaret H. Romano, and the Medical University of South Carolina/Hollings Cancer Center Tumor Bank for providing the lung tissue for these studies and their technical assistance. We thank Yusuf A. Hannun for his careful review of the manuscript.

	Footnotes

 
Received for publication December 21, 2004; accepted April 15, 2005

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