Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKα, -ε, and -ζ were detected in the lung. By immunohistochemical examination, DGKα and -ζ were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKα in the cytoplasm and DGKζ in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKα and -ζ was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKε was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.
- spatiotemporal expression patterns
the phosphoinositide (PI) cycle mediates one of the intracellular signal transduction pathways in eukaryotic cells and produces a class of second messengers that are involved in a variety of signaling cascades including cell growth, differentiation, hormonal and neurotransmitter action, and sensory perception. Triggering of the cell surface receptors, such as G protein-coupled receptors and receptor tyrosine kinases, initiates the cycle by activating phospholipase C (PLC), resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate into two second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3) (25). DG serves as an activator of phospholipid-dependent protein kinase C (PKC), whereas IP3 mobilizes Ca2+ from the endoplasmic reticulum (23). In addition to activating PKC, DG also targets other molecules, such as α- and β-chimaerins (having Rac-GTPase activating protein activity) (26) and guanyl nucleotide-exchange factors for Ras and Rap (16).
Diacylglycerol kinase (DGK) catalyzes phosphorylation of DG to generate phosphatidic acid (PA) and plays a major role in controlling the cellular level of DG (9, 33). Furthermore, there is accumulating evidence that PA, a product of DGK, also may serve as a second messenger that modulates the activity of several enzymes including PKC-ζ (20) and PLC-γ1 (15). Therefore, DGK occupies a central position in intracellular signal transduction through these two second messengers. DGK represents a large gene family of isozymes, and, in mammals, nine DGK isozymes have been isolated so far (9, 33). Previous studies have revealed that DGK isozymes show remarkable heterogeneity in structure, tissue expression, and enzymological property. We have so far reported the detailed cellular expression of mRNAs for the isozymes and their functional implications in the central nervous system and heart (5–8, 12, 32). These observations suggest that each isozyme has its own specific function in various biological processes.
The PI cycle has been shown in lung cells to mediate a variety of cellular signaling pathways (18, 22). In addition to the functional significance of DGK in signal transduction, it should also be mentioned that DG and PA are important intermediates for the synthesis of various lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine, and triacylglycerol, which are intimately involved in the biosynthetic pathway for surfactant in the lung (27). Therefore, we should take into consideration how DGK is involved in these apparently distinct mechanisms, i.e, surfactant synthesis and signal transduction.
To gain an insight into the functional implication of DGK in the lung, we first attempted to clarify the expression and localization pattern of DGK isozymes together with the developmental changes in their mRNA expressions and enzymatic properties in rat lung. Our results clearly show different localization of each DGK isozyme and its characteristic gene expression pattern in developing rat lung, suggesting that each DGK isozyme plays a different functional role in a different developing stage of the lung.
MATERIALS AND METHODS
This study was carried out in accordance with the Guide for Animal Experimentation, Yamagata University School of Medicine and the Law (no. 105) and Notification (no. 6) of the Government (approval no. 04-004). Wistar rats of various developmental stages were used in this study, including adults (7–8 wk), neonates (0–2 days), and embryos of defined gestational stages. For gestational staging, the morning of the first day after conception was regarded as the embryonic (E) day 0 (E0), and the day of birth as the postnatal day 0 (P0). Rats were anesthetized with pentobarbital (10 mg/kg body wt) and killed by decapitation. The organs were rapidly removed.
Northern blot analysis.
Total RNAs were extracted from whole rat lung at different stages of development and from adult rat brains (positive control) by acid guanidinium thiocyanate/phenol/chloroform extraction (TRIzol; GIBCO BRL, Bethesda, MD). Each of the total RNA samples (20 μg/lane) was denatured with formamide and size separated by formalin/agarose gel electrophoresis. The RNAs were transferred and fixed to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridized with the [32P]dATP-labeled probes for each rat DGK isozymes (α, β, γ, ε, ζ, and ι) as previously described (5, 6, 7, 8, 14, 17). Quantitative analysis of the signals was performed using a densitometer (Atto Densitograph; Atto, Tokyo, Japan), and the values were normalized for relative amounts of 18S ribosomal RNA.
First-strand cDNA was synthesized from 2 μg of RNA using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) following the manufacturer's instructions. PCR amplification was performed with KOD-plus polymerase (Toyobo, Tokyo, Japan) using gene-specific oligonucleotide primers for rat DGK isozymes as follows: DGKα forward, 5′-GTGACTGTGGACTGCTCCGTG-3′; DGKα reverse, 5′-CAACACAGCGACTGGAGGCAC-3′; DGKβ forward, 5′-GGACAGCATGTGTGGCGACTC-3′; DGKβ reverse, 5′-GTTCCGGCAGTGGGCATAGTC-3′; DGKγ forward, 5′-GTGGGATCCCACAGAGCTCAG-3′; DGKγ reverse, 5′-GACGGAGGAGTTCCCTTCCAC-3′; DGKε forward, 5′-CAAGGGCCTGGTACTGTCACC-3′; DGKε reverse, 5′-CCAAGGCAGTGCACAATGCGG-3′; DGKζ forward, 5′-CTGCCCCAAGGTGAAGAGCTG-3′; DGKζ reverse, 5′-GCTGTCTCCTGGTCCTCACGT-3′. PCR conditions were as follows: 95°C for 1 min; 30 cycles of 94°C for 30 s, 62°C for 30 s, 68°C for 40 s; and 68°C for 2 min. For normalization, rat GAPDH mRNA was simultaneously amplified using forward 5′-TTAGCACCCCTGGCCAAGG-3′ and reverse 5′-CCTACTCCTTGGAGGCCATG-3′ primers. PCR products amplified were separated by agarose gel electrophoresis, stained with ethidium bromide, and subjected to densitometric analysis as described above.
Excised lungs were endotracheally infused and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), followed by immersion in the same fixative for 2 h at 4°C. After cryoprotection in 30% sucrose in 0.1 M phosphate buffer, the tissue blocks were cut into 10-μm sections on a cryostat, placed on glass slides, and air-dried briefly. The sections were treated with 0.3% Triton X-100 in PBS for 2 h, followed by 0.3% H2O2 in methanol for 10 min at 4°C to inactivate endogenous peroxidase. They were then treated with 5% normal goat serum in PBS (NGS/PBS) to block nonspecific binding sites and incubated with anti-rat DGKα antibody (5) or anti-rat DGKζ antibody in NGS/PBS (12) overnight at 4°C in a moist chamber. Then, the sections were incubated with biotinylated goat anti-rabbit IgG (dilution 1:250; Vector Laboratories, Burlingame, CA) in NGS/PBS for 30 min at room temperature. The sites of antigen-antibody reaction were visualized by avidin-biotinylated peroxidase complex system (Vector Laboratories) with diaminobenzidine (Sigma) in 0.05 M Tris·HCl buffer (pH 7.6) containing 0.005% H2O2. The sections were dehydrated in graded alcohols, cleared with xylene, and mounted. For immunofluorescent staining, the sections were incubated with goat anti-rabbit IgG-Alexa 546 (dilution 1:400; Molecular Probes, Eugene, OR) in NGS/PBS for 30 min at room temperature instead of biotinylated goat anti-rabbit IgG. Some sections were further incubated with the antibody against SP-C (goat monoclonal, dilution 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) as a marker for alveolar type II cells or the antibody against ED-1 (mouse monoclonal, dilution 1:800; Pharmingen, San Diego, CA) as a marker for macrophages. Then, the sections were incubated with biotinylated horse anti-goat IgG (for anti-SP-C antibody, Vector Laboratories) or biotinylated goat anti-mouse IgG (for anti-ED-1 antibody, Molecular Probes), followed by streptavidin-Alexa 488 (dilution 1:250; Molecular Probes). Immunofluorescent image was observed under a fluorescent microscope (Leica Q550FW) and confocal laser-scanning microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany) at 543 nm helium excitation and 488 nm argon excitation and processed using Adobe Photoshop.
Isolation of rat alveolar type II cells and alveolar macrophages.
Alveolar type II cells were isolated from specific pathogen-free adult male Wistar rats by pancreatic elastase digestion and metrizamide density-gradient centrifugation, according to the method described by Dobbs and Mason (3). Alveolar macrophages were collected by bronchoalveolar lavage and used without further purification. These procedures yielded ∼98% purity for both cell types, which was determined by immunohistochemical staining for anti-SP-C or ED-1 antibody (data not shown). Isolated cells were placed on glass microscope slides by cytospin preparation and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min, and immunohistochemical staining was performed as described above. Some of the cells were used for enzymatic assay for DGK.
Protein extraction and DGK activity.
For measurement of DGK activity, total protein was extracted from adult rat brain, lung, isolated alveolar type II cells, and alveolar macrophages using lysis buffer. The homogenates were centrifuged at 14,000 g for 10 min to remove cell debris. Resulting supernatants were used for the assay. The protein concentration was determined by a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). DGK activity was measured by octylglucoside-mixed micelle assay using 1-stearoyl-2-linoleoyl-sn-glycerol (C18:0/C18:2 DG) (Biomol, Plymouth Meeting, PA) and 1-stearoyl-2-arachidonoyl-sn-glycerol (C18:0/C20:4 DG) (Biomol) as previously described (10). Assay was performed in the presence of each substrate at a concentration of 1 mM. The reaction mixture (50 μl) contained 50 mM MOPS (pH 7.2), 50 mM octylglucoside (Calbiochem, San Diego, CA), 100 mM NaCl, 1 mM dithiothreitol, 20 mM NaF, 2.1 mM CaCl2, 2.0 mM EGTA, 0.8 mM EDTA, 10 mM MgCl2, 6.7 mM phosphatidylserine (Avanti Polar Lipids, Birmingham, AL), and 1 mM [γ-32P]ATP (10,000 counts per minute/nmol; ICN Biomedicals, Costa Mesa, CA). The reaction was continued for 10 min at 30°C. Lipids were extracted and separated on thin-layer plates of silica gel (Merck, Darmstadt, Germany). The band of PA detected by autoradiography was scraped with a sharp spatula and collected for liquid scintillation counting. Under the conditions described above, the rate of the reaction was linear for at least 10 min.
Values are shown as means ± SD for each experimental group. Groups were compared using one-way analysis of variance and the Tukey-Kramer multiple-comparison tests for differences between groups. P < 0.05 was considered to be statistically significant.
Expression and localization of DGK isozymes in the lung.
In Northern blot analysis, hybridization signals for the three DGK isozymes (α, ε, ζ) were detected in adult rat lung (Fig. 1A). Positions of the hybridization bands for the three isozymes in the lung were the same as those in the brain. The expression levels based on the strength of the hybridization bands of DGKα and -ζ were high, whereas that of DGKε was low. RT-PCR analysis also confirmed the expression profile of the DGK isozymes in the lung described above (Fig. 1B).
To examine which cells were responsible for the expression, we performed immunohistochemical analysis of the lung using specific antibodies against rat DGKα and -ζ (Figure 2). Immunoreactivity for DGKα was recognized in round alveolar cells located near the angles between neighboring alveolar septa. Some were located in the alveolar space, suggesting that alveolar macrophages are also immunoreactive. There was no immunoreactivity detected in other regions including bronchus, trachea, and mesothelium. On the other hand, immunoreactivity for DGKζ was detected in the nucleus of various types of epithelial cells, such as bronchus, trachea, and mesothelium in addition to the round cells in alveoli, showing much broader expression of DGKζ than that of DGKα. These immunohistochemical data strongly suggest that both alveolar type II cells and macrophages are responsible for the expression of DGKα and -ζ.
Double-immunofluorescent staining of alveolar type II cells and macrophages.
To confirm the expression of DGKα and -ζ in alveolar type II cells and macrophages, we performed double-immunofluorescent analysis using specific markers for those cells, i.e., anti-SP-C antibody and anti-ED-1 antibody for alveolar type II cells and macrophages, respectively. As shown in Fig. 3A, most of the DGKα-immunoreactive cells were also SP-C immunoreactive, indicating that DGKα is expressed in alveolar type II cells. On the other hand, DGKζ-immunoreactive cells outnumbered SP-C-immunoreactive cells in the alveoli (Fig. 3B), suggesting that DGKζ is expressed not only in type II cells but also in type I cells, although at low levels. With regard to alveolar macrophages, ED-1-immunoreactive macrophages were also immunoreactive for both DGKα and -ζ, showing that these isozymes are expressed in these cells (Fig. 4).
Expression of DGKα and -ζ in isolated alveolar type II cells and macrophages.
We further examined the expression of DGKα and -ζ using isolated alveolar type II cells and macrophages. The isolation procedures yielded ∼98% purity for both cell types, which was determined by immunocytochemical staining for anti-SP-C or anti-ED-1 antibody (data not shown). Immunoreactivities for DGKα and -ζ were clearly detected in both cell types, with the former fine dotted in the cytoplasm and the latter speckled in the nucleus (Fig. 5). These results were the same as those of the immunostaining in the lung, further confirming that DGKα and -ζ are expressed in alveolar type II cells and macrophages.
DGK activity in the whole lung, alveolar type II cells, and macrophages.
In the previous study, DGK activity has been detected in rat lung (13). However, recent molecular cloning studies of several DGK isozymes have revealed different enzymological properties of the isozymes, i.e., DGKα, -β, and -γ are activated by Ca2+, and DGKε specifically phosphorylates arachidonoyl-containing DG (1-stearoyl-2-arachidonoyl-sn-glycerol) (33). Therefore, we reexamined DGK activity in the whole lung with attention to Ca2+ dependency and substrate specificity. We also examined the enzymatic activity of the isolated alveolar type II cells and macrophages. An octylglucoside-mixed micelle assay was used to evaluate substrate specificity toward 1-stearoyl-2-linoleoyl-sn-glycerol (18:0/18:2 DG) and 1-stearoyl-2-arachidonoyl-sn-glycerol (18:0/20:4 DG) in the presence of 0.1 mM Ca2+ (21). As shown in Fig. 6A, DGK activity was detected in the whole lung at 10-fold lower levels in the brain where most of the isozymes are abundantly expressed. The activities toward 18:0/18:2 DG and 18:0/20:4 DG were almost comparable in the lung, showing no apparent substrate specificity. DGK activities in the isolated alveolar type II cells and macrophages were ∼20% and 150% compared with that of the whole lung, respectively, and almost equal toward 18:0/18:2 DG and 18:0/20:4 DG. In the presence (0.1 mM) or absence of Ca2+, the whole lung and the isolated cells showed similar enzymatic activities (Fig. 6B).
Expression of DGK isozymes in the developing lung.
Development of the lung begins on approximately E11 in rats as a ventral foregut outpouching. Subsequently, repetitive branching of the lung bud occurs with eventual formation and maturation of the alveolar acinar units, together with specialized epithelial cell differentiation and surfactant production. In the perinatal period, the sudden exposure of the air-blood interface of the lung to increased oxygen tension must cause drastic changes to lung cells. To understand the functional implications of DGK isozymes during lung development, we examined temporal expression patterns of mRNA for DGK isozymes in the developing lung by Northern blot analysis using 18S ribosomal RNA as a control (Fig. 7A). Hybridization signals for DGKα showed a constant level during the embryonic stage but gradually increased after birth. At adult stage, the signals were approximately fourfold higher than those of the embryonic stage and almost equivalent to those of adult brain. On the other hand, the hybridization signals for DGKε were nearly constant during embryonic and postnatal stages. A similar expression pattern was found in DGKζ except that the signals increased slightly after birth.
It should be noted, however, that during the perinatal period, distinct expression patterns were observed for DGKα, -ε, and -ζ (Fig. 7A): the expression of DGKε was transiently elevated immediately after birth (P0) compared with that on E21. On the other hand, the expression for DGKα and -ζ was inversely elevated on E21 and then decreased on P0. To further examine these changes during the perinatal period, we used semiquantitative RT-PCR with GAPDH as a control (Fig. 7B). The data clearly showed that the expression for DGKε was transiently elevated approximately fourfold on P0 compared with that on E21 and then decreased on P1, although changes in the expression for DGKα and -ζ during the perinatal period were not statistically significant. These data suggest that DGK isozymes play distinct roles in developing lung, especially during the perinatal period.
Although DGK activity has previously been detected in rat lung (10), the present study reports for the first time detailed information on the expression and localization of DGK isozymes in the lung. Recent studies have identified several DGK isozymes, which show remarkable heterogeneity in terms of gene structure, tissue expression, and enzymological properties. Such advances raise a new question: which isozyme(s) of DGK is responsible for the physiological and pathological function in the lung? Here we identify the gene expression of DGKα, -ζ, and -ε in this organ. Furthermore, by immunohistochemical examination using currently available antibodies against DGKα and -ζ, both isozymes are coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes are localized at distinct subcellular locations, i.e., DGKα in the cytoplasm and DGKζ in the nucleus, suggesting different roles for these isozymes.
In terms of functional implications, it is reported that DGKα is detected in oligodendrocytes of the brain and T lymphocytes (5) and that IL-2 receptor is one of the molecules commonly expressed in both of these cell types (24). IL-2 is shown to have some effects not only on the proliferation of T cells but also on the functions of oligodendrocytes, such as the proliferation, differentiation, and regulation of myelin proteins (1, 4, 30). In addition, recent reports have shown that IL-2 receptor is also expressed in alveolar type II cells and macrophages and that the expression of IL-2 receptor is enhanced in alveolar macrophages during inflammation induced by interferon-γ (10, 19). In this regard, it is of particular importance that IL-2-induced T cell proliferation may be mediated through PA generated by DGKα. (4). Given that the molecular machinery that propagates the IL-2-mediated signal transduction may be shared among relevant cells in general, the expression of DGKα in these lung cells, as revealed in this study, suggests that this isozyme might be involved in this pathological process in the lung, which warrants further investigation.
Nuclear localization of DGKζ in the lung cells is compatible with our previous study of its immunohistochemical examination in neurons (12), which together with this study, indicates that DGKζ is localized in the nuclei of both proliferating and nonproliferating cells. Although the functional role applicable to all kinds of DGKζ-expressing cells is unclear, our previous study of myocardial infarction model of rats shows that the mRNA expression for DGKζ is enhanced in macrophages that are infiltrated into the necrotic area, suggesting the possible role of DGKζ in phagocytosis of these cells (32). It remains to be elucidated whether macrophages intrinsically express DGKζ at a high level or whether the increased expression is induced by the activation of the phagocytic reaction.
One of the best-characterized functional roles of DGK is in the regulation of PKC, for which DG acts as an allosteric activator (23). In this regard, there is evidence that PKC may play an important role in the regulation of surfactant secretion in type II cells (28, 29). Agonists that activate PKC, such as 12-O-tetradecanoylphorbol-13-acetate and cell-permeable DGs, are shown to serve as the most effective surfactant secretagogues in isolated type II cells (29). Rooney et al. (28) describes that ATP triggers the PI cycle via P2Y2 purinoceptors, which results in the activation of PKC and subsequent stimulation of surfactant secretion. Furthermore, calphostin C, a potent inhibitor of PKC, blocked PC secretion stimulated by glucagon-like peptide 1, the truncated and amidated form of glucagon-like peptide 1, in human alveolar type II cells (34). Thus it is highly plausible that DGK may play a role in regulating surfactant secretion, although which one is responsible for the regulation among the three isozymes identified in this study remains to be elucidated.
Enzymatic assays for the whole lung, isolated alveolar type II cells, and macrophages reveal that total DGK activities in these samples show neither Ca2+ dependency nor substrate specificity. To date, enzymatic property has been well characterized for DGK isozymes: Ca2+-dependent activation for DGKα, -β, and -γ, and specific phosphorylation toward arachidonoyl-containing DG for DGKε (33). Considering the present Northern blot and RT-PCR data showing that DGKα, -ζ, and -ε are responsible for the expression in the lung, DGKζ, a Ca2+-independent isozyme with no substrate specificity, may be dominant among those in terms of the activity.
It should also be mentioned that DGK isozymes show unique patterns of expression during the perinatal period. Using semiquantitative RT-PCR, we found that the expression for DGKε was transiently elevated at approximately fourfold immediately after birth (P0) during the perinatal period. Lung is one of the organs exposed to drastic changes before and after birth. In this regard, the biochemical adaptation of the lung to air or oxygen breathing at birth is incompletely understood. A sudden exposure of the air-blood interface of the lung to increased oxygen tension must pose an acute oxidative stress compared with the relatively anaerobic fetal environment. Indeed, the pulmonary epithelium is usually exposed to the highest oxygen tension present in the organism (2). Our data show that DGKε is very unique in that its expression is upregulated by increased oxygen tension, whereas expression for DGKα and -ζ is downregulated. It is known that PI has a characteristic fatty acid composition of 1-stearoyl-2-arachidonoyl (11). Considering the substrate specificity of DGKε toward arachidonoyl-DG, a possible link might be suggested between oxygen stress and PI metabolism involved with DGKε. This hypothesis may be partly consistent with the previous report that reactive oxygen species, such as hydrogen peroxide, activate several enzymes involved in lipid signaling, such as PI-specific PLC, in several cultured cell types (31). Further studies are needed to clarify this point.
In conclusion, our results reveal for the first time the gene expression of DGKα, -ζ, and -ε in the lung. Furthermore, immunohistochemical analysis shows that DGKα and -ζ are coexpressed in alveolar type II cells and macrophages and that these isozymes are localized at distinct subcellular locations. In the developing lung, distinct expression patterns for DGKα, -ε, and -ζ are observed during the perinatal period. All these data suggest that each isozyme plays a different role in lung functions such as surfactant production and secretion, phagocytic reaction, and adaptation to oxygen breathing. Identification of specific isozymes of DGK in the lung would help us further investigate detailed physiological and pathological roles for each molecule.
This work was supported by Grant-in-Aid and the 21st Century of Excellence Program from the Ministry of Education, Science, Culture, and Sports of Japan (I. Kubota and K. Goto) and by the Ono Medical Research Foundation, Kato Memorial Bioscience Foundation, and Janssen Pharmaceutical (K. Goto).
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