Cell Biology International 31 (2007) 148e155www.elsevier.com/locate/cellbi

Differential expression of phospholipases D1 and D2 in mouse tissues

Heechul Kim a, Jeeyoung Lee a, Seungjoon Kim a, Min Kyoung Shin b,Do Sik Min b,*, Taekyun Shin a,**

a Department of Veterinary Medicine, Cheju National University, Jejudaehakro 66, Aradong, Jeju city, Jeju-do 690-756, South Koreab Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, South Korea

Received 11 July 2006; revised 16 August 2006; accepted 26 September 2006

Abstract

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues,including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus.

In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. BothPLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach,colon, kidney, testes, and uterus.

Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLDimmunostaining in the liver, kidneys, spleen, and heart.

These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme playsa distinct role in each organ.� 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Phospholipase D; Immunoprecipitation; Tissue distribution; Mouse

1. Introduction

Phospholipase D (PLD) catalyzes the hydrolysis of phos-phatidylcholine, which is the major membrane phospholipid,to form phosphatidic acid (PA) and choline (Exton, 1998,1999). PA can alter the activities of many enzymes and pro-teins (Andresen et al., 2002) and can be further metabolizedto diacylglycerol and lysophosphatidic acid by PA phosphohy-drolase and phospholipase A2, respectively. Therefore, PLDinfluences many important intracellular events via the

Abbreviations: CNS, central nervous system; FITC, fluorescein isothiocya-

nate; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei; PA, phos-

phatidic acid; PKC, protein kinase C; PLD, phospholipase D; TRITC,

tetramethyl rhodamine isothiocyanate.

* Corresponding author. Tel.: þ82 51 510 3682; fax: þ82 51 513 9258.

** Corresponding author. Tel.: þ82 64 754 3363; fax: þ82 64 756 3354.

E-mail addresses: minds@pusan.ac.kr (D.S. Min), shint@cheju.ac.kr

(T. Shin).

1065-6995/$ - see front matter � 2006 International Federation for Cell Biolog

doi:10.1016/j.cellbi.2006.09.020

production of these downstream products. PLD activity isregulated by many stimuli, such as cytokines, growth factors,hormones, neurotransmitters, and other molecules that are in-volved in extracellular communication (Exton, 1998). PA,which is generally recognized as the signaling product ofPLD, functions as an effector in multiple physiological pro-cesses, including cell proliferation, differentiation, secretion,and migration (Boarder, 1994; Exton, 1998, 1999).

In mammals, two isoforms of PLD, PLD1 and PLD2, havebeen cloned and characterized for their regulatory and cellularfunctions (Frohman and Morris, 1999). The activation of PLDoccurs through interactions between the ADP-ribosylationfactor and Rho families, as well as protein kinase C (PKC)(Frohman and Morris, 1999).

A previous study reported significant activities for the Rho-and ADP-ribosylation factor-responsive forms of PLD in themembranes of all tissues, with the exception of muscle, withparticularly high levels in the lungs, spleen, and kidneys ofrats (Provost et al., 1996) and in the central nervous system

y. Published by Elsevier Ltd. All rights reserved.

149H. Kim et al. / Cell Biology International 31 (2007) 148e155

(CNS) tissues of rats (Lee et al., 2000a,b, 2001). In addition tothe constitutive expression of PLD in CNS tissues, PLD isknown to be consistently activated in the macrophages presentin inflammatory lesions, including those associated with cere-bral cryoinjury (Kim et al., 2004), spinal cord injury (Junget al., 2003), experimental autoimmune encephalomyelitis(Ahn et al., 2001), and autoimmune neuritis (Shin et al.,2002), as well as those found in the heart during autoimmunemyocarditis (Ahn et al., 2004). Although PLD expression hasbeen studied in various organs under pathological conditions,as described above, little is known about the differential ex-pression patterns of PLD1 and PLD2 or the cellular localiza-tions of PLD in the various tissues of laboratory animals,including mice.

The aim of this study was to examine the expression and cel-lular localization of PLD in various murine tissues, i.e. the cere-brum, cerebellum, heart, lung, liver, spleen, stomach, pancreas,ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus,using Western blot analysis and immunohistochemistry.

2. Materials and methods

2.1. Animals

Five male and five female BALB/c mice (25 g, 8e10 weeks of age) were

used in this experiment. Tissues from both male and female mice were used

for the Western blot analysis, while four mice were used for the histology

(n¼ 3e5 samples per gender). All of the experiments were carried out in

accordance with the National Research Council’s Guide for the Care and

Use of Laboratory Animals.

2.2. Tissue preparation

Various murine tissues, which included the cerebrum, cerebellum, heart,

lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys,

testes, ovary, and uterus, were dissected and lysed in immunoprecipitation

buffer (20 mM HEPES (pH 7.2), 1% Triton X-100, 1% deoxycholate,

0.1% SDS, 150 mM NaCl, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM

phenylmethylsulfonylfluoride).

Pieces of tissue from the cerebrum, cerebellum, heart, lung, liver, pancreas,

kidney, spleen, intestine, adrenal gland, testis, ovary, and uterus, were fixed in

4% paraformaldehyde in phosphate buffer and embedded in paraffin. Paraffin

sections were stained with hematoxylin and eosin, and these sections were also

used for immunohistochemistry.

2.3. Antisera

Antisera were raised against the C-terminal peptide of PLD1, which corre-

sponds to amino acid residues 1063e1074 (TKEAIVPMEVWT) of the rat

PLD1 sequence. The antisera reacted with both PLD1 and PLD2. For affinity

purification of the antibodies, the peptide was coupled to Affi-Gel 15 (Bio-

Rad, Hercules, CA, USA) according to the manufacturer’s instructions with

a slight modification. The anti-PLD1 antibody raised against the C-terminal

12 amino acid residues of PLD1 recognized PLD2 because the C-terminal

seven amino acids of PLD2 and PLD1 are identical.

The anti-PLD antibody used in this study has been shown previously to

label reactive astrocytes and macrophages in rats with EAE (Ahn et al.,

2001), ischemic brain injury (Lee et al., 2000b), and clip compression injury

(Jung et al., 2003), as well as ganglion cells and glial cells, including Muller

cells, in the developing and adult retina of the rat (Lee et al., 2001, 2002).

PLD1 and PLD2 were co-transfected into U87 MG glioma cells, and extracts

of the transfected cells were used as control samples that contained PLD1

and PLD2.

2.4. Immunoprecipitation and Western blotting

Tissue samples were homogenized in immunoprecipitation buffer. After

incubation for 30 min in an ice-bath, the homogenates were centrifuged, and

the resulting cell lysates were spun at 15,000�g in an Eppendorf Microcentri-

fuge for 10 min at 4 �C, to pellet the unbroken cells. The protein concentra-

tions of the supernatants were determined using the Bradford method, with

bovine serum albumin as the standard. The lysate supernatant was precleared

by incubation with preimmune IgG and protein A-Sepharose for 30 min. Pre-

cleared cell lysates were incubated with the anti-PLD antibody and 30 ml of

the 50% slurry of protein A-Sepharose for 4 h. The immune complex was col-

lected by centrifugation and washed five times with ice-cold buffer that con-

tained 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl,

2 mM Na3VO4, 10% glycerol, and 1% Nonidet P-40. SDS sample buffer

was added and the mixture was boiled. The recovered proteins were separated

by SDS-polyacrylamide gel electrophoresis. The separated proteins were

transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore,

Billerica, MA, USA), blocked in 5% non-fat dried milk, and probed with the

anti-PLD antibody as indicated. Immunoreactive bands were visualized by

chemiluminescence using horseradish peroxidase-conjugated secondary anti-

bodies and the ECL reagents (Amersham Biosciences, Piscataway, NJ, USA).

2.5. Immunohistochemistry

Paraffin sections (5-mm thickness) were deparaffinized and pretreated with

0.01 M citrate buffer (pH 6.0) in a microwave oven for 3 min. After hydration,

the sections were treated with 0.3% hydrogen peroxide in distilled water for

20 min, to block endogenous peroxidase activity. Following three washes in

PBS, the sections were exposed to 10% normal goat serum and allowed to re-

act with the affinity-purified anti-PLD antibody. Immunoreactivity was visual-

ized with the avidinebiotin peroxidase reaction (Vector Elite ABC kit; Vector

Labs, Burlingame, CA, USA). Peroxidase was developed with diaminobenzi-

dine substrate kit (Vector Labs). The sections were counterstained with hema-

toxylin before being mounted. As a negative control, the primary antisera were

omitted from few test sections in each experiment; no specific labeling was

detected in these sections.

To examine the cell phenotype of PLD expression, double immunofluores-

cence was applied using cell type-specific markers, including anti-neuronal

nuclei (NeuN) antibody (Chemicon, Temecula, CA, USA) for neurons, anti-glial

fibrillary acidic protein (GFAP) antibody (Sigma, St. Louis, MO, USA) for

astrocytes, and anti-insulin antibody (Sigma) for b-cells of the pancreatic islets

of Langerhans. In brief, the sections were incubated in the following order:

10% normal goat serum for 1 h at RT, rabbit anti-PLD antibody overnight at

4 �C, and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG

(Sigma) for 1 h at RT. The sections were then washed and blocked with 10% nor-

mal goat serum for 1 h at RT, incubated overnight at 4 �C with either the mouse

monoclonal anti-NeuN or anti-GFAP antibody for the brain sections or the anti-

insulin antibody for the pancreas sections. After three washes, the slides were

reacted with tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-

mouse IgG (Sigma). The double immunofluorescence-stained specimens were

examined with the FV500 laser confocal microscope (Olympus, Tokyo, Japan).

To reduce or eliminate lipofuscin autofluorescence, the sections were

washed three times for 1 h in PBS at RT, dipped briefly in distilled H2O,

treated with 10 mM CuSO4 in ammonium acetate buffer [50 mM

CH3COONH4 (pH 5.0)] for 20 min, dipped briefly again in distilled H2O,

and then placed in PBS. The double immunofluorescence-stained specimens

were examined with the FV500 laser confocal microscope (Olympus).

3. Results

3.1. Differential expression patterns ofPLD1 and PLD2 in mouse tissues

To investigate the differential expression patterns of PLDisozymes in diverse tissues of the mouse, lysates of varioustissues were immunoprecipitated and analyzed by Western

150 H. Kim et al. / Cell Biology International 31 (2007) 148e155

Fig. 1. Western blot analysis of PLD1 and PLD2 expression in various tissues of the mouse. The tissue homogenates were immunoprecipitated with anti-PLD

antibody and immunoblotted with anti-PLD antibody. The anti-PLD1 antibody specifically recognizes PLD1 and PLD2 in various mouse tissues. The line on

the left indicates the molecular size markers. Standard indicates control samples of both PLD1 and PLD2, which were prepared by extracting cells’ glioma after

co-transfection of PLD1 and PLD2 cDNA.

blotting. The anti-PLD antibody used in this study was raisedagainst the C-terminal 12 amino acid residues of PLD1; thisantibody recognizes PLD2, as the C-terminal seven aminoacids of PLD1 and PLD2 are identical.

As shown in Fig. 1, the anti-PLD1 antibody specificallyrecognized the PLD1 (approximately 120 kDa) and PLD2 (ap-proximately 105 kDa) control samples, which were extractedfrom U87 MG astroglioma cells that were co-transfected

Fig. 2. Immunohistochemical localization of PLD in the cerebrum (A, E, H) and cerebellum (B). PLD immunoreactivity is visualized in some neurons (A, arrows)

of the cerebral cortex. In the cerebellum, Purkinje cells (B, arrows) are intensely immunopositive for PLD. Some NeuN-positive neurons (F, red arrows) are im-

munoreactive for PLD (E, green arrows) in the cerebrum (G, merged arrows). PLD (H, green arrow) and GFAP (I, red arrow) were co-localized in the cerebrum in

some astrocytes (J, merged arrow). No specific reaction product is seen in the cerebrum (C) or cerebellum (D) with the use of non-immune sera. (AeD) Counter-

stained with hematoxylin. Scale bars¼ 30 mm.

151H. Kim et al. / Cell Biology International 31 (2007) 148e155

with the PLD1 and PLD2 cDNAs. Only PLD1 was detected inthe heart and ovary, while only PLD2 was detected in the pan-creas and ileum. Both PLD1 and PLD2 were strongly ex-pressed in the cerebrum, cerebellum, and lung, while bothPLD1 and PLD2 were expressed in the liver, spleen, stomach,colon, kidney, testis, and uterus. Neither PLD1 nor PLD2 wasdetected in the adrenal gland.

3.2. Localization of PLD in various tissues of the mouse

In the central nervous system tissues, PLD was immuno-stained in many of the neurons in the cerebral cortex of the ce-rebrum (Fig. 2A). In the cerebellum, PLD was stronglyimmunostained in the Purkinje cells, but was not detected inthe granular cells of the granular cell layer (Fig. 2B). Further-more, high levels of PLD were immunodetected in NeuN-positive neurons (Fig. 2EeG) in the cerebral cortex. PLD wasimmunodetected in some GFAP-positive astrocytes in the cere-brum, including the hippocampal dentate gyrus (Fig. 2HeJ).

In the heart, PLD was weakly immunostained in some car-diomyocytes (Fig. 3A). The lungs showed intense PLD immu-noreactivity in the covering epithelia of the bronchioles and insome macrophages, as well as in the pneumocytes of the al-veoli (Fig. 3B).

In the gastrointestinal tract (Fig. 4AeC), PLD was consti-tutively detected in some of the cells in the gastric glands,as well as in the epithelial cells of the small intestine (ileum)

and large intestine (colon). In the ileum, PLD was intenselyimmunostained in the cells of the crypts of the intestinalglands of the small intestine. In the liver, PLD was weakly im-munostained in the hepatocytes and in some of the epithelia ofthe bile ducts (Fig. 4E).

In the pancreas, PLD was immunostained in the pancreaticislets and in the epithelia of the pancreatic ducts. PLD wasalso weakly immunostained in the pancreatic acini (Fig. 4Fand J). Furthermore, PLD immunoreactivity was co-localizedwith insulin-positive b-cells of the pancreatic islets of Langer-hans (Fig. 4JeL). This suggests that PLD is closely associatedwith the b-cells of the pancreatic islets of Langerhans.

In the kidneys, PLD was immunostained in the proximaland distal tubules in the cortex, but was scarcely detected inthe renal medulla, collecting tubules, and glomeruli (Fig. 5Aand B). In the urinary bladder, PLD was immunostained inthe covering transitional epithelium (Fig. 5D).

In the adrenal gland, PLD immunostaining was scarce inthe adrenal cortex and medulla (data not shown), and in thespleen, some PLD-positive cells were scattered throughoutthe red pulp (data not shown).

In the testes, PLD was stained, albeit weakly, in the sper-matogonia and Sertoli cells, and was intensely stained in thespermatocytes and residual bodies in the spermatids of theseminiferous tubules. PLD was scarcely detected in the sper-matozoa in this study. PLD was intensely immunostained inthe Leydig cells in the interstitial spaces (Fig. 6A).

Fig. 3. Immunohistochemical localization of PLD in the heart (A) and lung (B). In the heart, some cardiomyocytes are weakly immunoreactive for PLD (A,

arrows). In the lung, PLD is localized in the covering epithelia of the bronchioles (arrowheads) and some macrophages (arrows), as well as in the pneumocytes

of the alveoli (B). No specific reaction product is seen in the heart (C) or lung (D) with the use of non-immune sera. Counterstained with hematoxylin. Scale

bars¼ 30 mm.

152 H. Kim et al. / Cell Biology International 31 (2007) 148e155

Fig. 4. Immunohistochemical localization of PLD in the stomach (A, B), small intestine (C), liver (E), and pancreas (F, J). In the stomach, PLD immunoreactivity is

intense in the covering epithelial cells (A, arrows) and chief cells (B, arrows), and weak in the parietal cells (B, arrowheads). In the small intestine, some epithelial

cells and glands (arrows) are immunopositive for PLD (C). In the liver, PLD is weakly immunostained in some hepatocytes and in the epithelia of bile ducts (E,

arrows). In the pancreas, PLD immunoreactivity is intense in the pancreatic islets (asterisk) and epithelium of the pancreatic ducts (arrows) and moderate in the

pancreatic acini (arrowheads) (F, J). In the double-labeling experiment, PLD (J, green asterisk) is co-expressed in an insulin-rich area (K, red asterisk) of the pan-

creatic islets (L, merged asterisk). No specific reaction product is seen in the stomach (D), small intestine (G), liver (H) or pancreas (I) with the use of non-immune

sera. (AeI) Counterstained with hematoxylin. Scale bars¼ 30 mm.

In the ovary, PLD was immunostained in the granulosa cellsand theca cells during various stages of the growing follicles, inthe oocytes in the primordial and unilaminar follicles, and instromal cells (Fig. 6B). In the uterus, PLD was immunostainedin the cuboidal epithelium and endometrial glands (Fig. 6C).

4. Discussion

This study confirms the differential expression of PLD1and PLD2 in various organs of the mouse, and represents

the first description of the cellular localization of PLD invarious tissues of the mouse.

Although two isoforms of PLD, PLD1 and PLD2, havebeen cloned and are being characterized in terms of cellularregulation and function in mammals, the functional differ-ences between the two PLD isoforms in cellular responsesare still poorly understood. Most previous studies on PLDhave been limited to the analyses of cultured cells to elucidatethe involvement of PLD in transmembrane signaling processes(Provost et al., 1996; Min et al., 1998, 2001a). It has been

153H. Kim et al. / Cell Biology International 31 (2007) 148e155

reported that PLD is expressed in a variety of cell types, suchas neurons, hematopoietic cells, Leydig cells, oocytes, parotidacini, spermatozoa, pancreatic acini, pancreatic islets, andpneumocytes (Exton, 1999).

Recently, the expression of PLD was confirmed in CNS tis-sues, which include the brain (Lee et al., 2000a,b), retina ofthe rat (Lee et al., 2001, 2002), and retina of the pig (Leeet al., 2006). Furthermore, PLD has been detected in heartswith autoimmune myocarditis (Ahn et al., 2004) and in thekidneys of rats with spontaneous hypertension (Min et al.,2001b). These findings suggest that PLD is expressed consti-tutively in various tissues, such as the heart and kidneys, aswell as in nerve tissues, which implies that PLD plays a rolein most mammalian organs.

Although PLD is expressed in most organs of the mouse, asobserved by measurements of PLD mRNA (Meier et al.,1999), the discussion of cellular localization of PLD in ourstudy is limited to the brain, testis, lungs, and intestine becausethese organs strongly express PLD, as compared to other tis-sues. This self-imposed limitation does not in any way dimin-ish the potential importance of PLD in other organs.

The expression of PLD in the cerebrum and cerebellum ofmice mirrors the situation in rats (Lee et al., 2000a), whichsuggests that PLD plays an important role in membrane sig-naling in CNS tissues.

In the testis, PLD was detected in the Leydig cells, as shownin previous studies (Vinggaard and Hansen, 1991; Lauritzen andHansen, 1995; Strand et al., 1999). This suggests that PLD is as-sociated with the production of hormones, such as testosterone.In the present study, in addition to the immunostaining of PLD

in Leydig cells, PLD localized weakly to the spermatogonia andSertoli cells, and was strongly detected in the spermatocytes andresidual bodies of spermatids in the seminiferous tubules, whilethere was little PLD in the spermatozoa. Given the presence ofPLD in the seminiferous tubules of mice, it appears that PLDplays an important role in spermatogenesis, particularly inspermiogenesis.

The nuclear staining of PLD in spermatocytes suggests thatPLD translocates into the nucleus when spermatocytes beginto divide. Recent studies have shown that nuclear RhoA-and ARF-activated PLD are present in the nuclear envelope(Baldassare et al., 1997; Banno et al., 1997). In addition,PLD1 has been immunolocalized in the nuclei of GH3 pitui-tary cells (Freyberg et al., 2001) and PLD2 has been detectedin human renal cancer cells (Zhao et al., 2000). These findingssuggest that the intranuclear activation of PLD plays importantroles in the proliferation of cancer cell lines and in spermato-genesis. The functional role of PLD in these processes needsto be investigated in future studies.

In the lungs, PLD was detected in the epithelia of the bron-chioles, which suggests that much of the covering epitheliumis proliferating in order to renew the epithelium. The localiza-tion of PLD in macrophages is a consequence of activation, asseen in autoimmune disease models (Ahn et al., 2004). SincePKC proteins, in particular the alpha and delta isoforms, areexpressed in the lungs (Wetsel et al., 1992), we postulatethat both PLD and PKC play roles in signaling in pulmonarytissues, including bronchiolar epithelia and alveoli. Moreover,it is clear that PLD is associated with the activation (or prolif-eration) of alveolar macrophages in mice.

Fig. 5. Immunohistochemical localization of PLD in the kidneys (A, B) and urinary bladder (D). In the kidneys, PLD immunoreactivity is more intense in the

cortex than in the medulla (A). In addition, the PLD immunoreactivities in the proximal and distal tubules (arrow) are more intense than those in the collecting

tubules (arrowhead) and renal corpuscles (RC) of the kidney (B). In the urinary bladder (D), PLD immunoreactivity is detected in the covering transitional

epithelium. No specific reaction product is seen in the kidney (C) or urinary bladder (E) with the use of non-immune sera. Counterstained with hematoxylin. Scale

bars: A, 300 mm; BeE, 30 mm.

154 H. Kim et al. / Cell Biology International 31 (2007) 148e155

Fig. 6. Immunohistochemical localization of PLD in the testis (A), ovary (B), and uterus (C). In the testis, PLD immunoreactivity is weak in the spermatogonia and

Sertoli cells, and intense in the spermatocytes (arrowheads) and residual bodies of the spermatids (arrows) in the seminiferous tubules, with little reactivity in the

spermatozoa (A). Furthermore, some Leydig cells (asterisks) in the interstitial space are strongly immunostained for PLD (A). In the ovary, PLD immunoreactivity

is detected in some oocytes in the primordial and unilaminar follicles (arrowheads), granulosa cells (asterisk), theca cells (arrows) in the growing follicles, and

stromal cells (B). In the uterus, the covering epithelium of the uterine mucosa (arrowheads) and endometrial glands (arrow) are immunopositive for PLD (C).

No specific reaction product is seen in the testis (D), ovary (E) or uterus (F) with the use of non-immune sera. Counterstained with hematoxylin. Scale

bars¼ 30 mm.

We found that the small intestine and large intestine haddifferent patterns of PLD expression in mice. Both PLD1and PLD2 were clearly visualized in the large intestine (co-lon), while only PLD2 was detected in the small intestine (il-eum). This differential expression phenomenon requiresfurther study.

In the rat pancreas, it has been reported that PLD1 is ex-pressed in the MIN6 pancreatic b-cells, and it is known to reg-ulate the secretion of insulin from pancreatic b-cells (Hugheset al., 2004). In the present study, we detected PLD2 exclu-sively in the mouse pancreas using immunoblot analysis,and PLD was intensively localized in the insulin-positive pan-creatic b-cells using immunohistochemistry. These findingssuggest that PLD2 plays a role in the mouse pancreas, partic-ularly in the pancreatic islets of Langerhans. To determine thespecies differences associated with PLD in the pancreas, weperformed Western blot analysis of PLD in rat pancreas, anddetected both PLD1 and PLD2 (data not shown), which sug-gests that PLD1 and PLD2 are differentially expressed in thepancreatic tissues of rats and mice.

Considering all of these findings, we postulate that PLD isexpressed constitutively in a variety of mouse organs. The roleof PLD in individual murine organs needs to be examined fur-ther in the future.

Acknowledgments

This work was supported by a program of the Basic AtomicEnergy Research Institute (BAERI), which is a part of the Nu-clear R & D Programs funded by the Ministry of Science &Technology (MOST) of Korea. This work was supported byPusan National University Research Grant, 2004.

References

Ahn M, Min DS, Kang J, Jung K, Shin T. Increased expression of phospholi-

pase D1 in the spinal cords of rats with experimental autoimmune enceph-

alomyelitis. Neurosci Lett 2001;316:95e8.

Ahn M, Lee Y, Sim K, Min DS, Matsumoto Y, Wie M, et al. Increased expres-

sion of phospholipase D in the heart with experimental autoimmune myo-

carditis in Lewis rat. Immunol Invest 2004;33:97e107.

Andresen BT, Rizzo MA, Shome K, Romero G. The role of phosphatidic acid

in the regulation of the Ras/MEK/Erk signaling cascade. FEBS Lett 2002;

531:65e8.

Baldassare JJ, Jarpe MB, Alferes L, Raben DM. Nuclear translocation of

RhoA mediates the mitogen-induced activation of phospholipase D involv-

ed in nuclear envelope signal transduction. J Biol Chem 1997;272:4911e4.

Banno Y, Tamiya-Koizumi K, Oshima H, Morikawa A, Yoshida S, Nozawa Y.

Nuclear ADP-ribosylation factor (ARF)- and oleate-dependent phospholi-

pase D (PLD) in rat liver cells. Increases of ARF-dependent PLD activity

in regenerating liver cells. J Biol Chem 1997;272:5208e13.

155H. Kim et al. / Cell Biology International 31 (2007) 148e155

Boarder MR. A role for phospholipase D in control of mitogenesis. Trends

Pharmacol Sci 1994;15:57e62.

Exton JH. Phospholipase D. Biochim Biophys Acta 1998;1436:105e15.

Exton JH. Regulation of phospholipase D. Biochim Biophys Acta 1999;1439:

121e33.

Freyberg Z, Sweeney D, Siddhanta A, Bourgoin S, Frohman M, Shields D. In-

tracellular localization of phospholipase D1 in mammalian cells. Mol Biol

Cell 2001;12:943e55.

Frohman MA, Morris AJ. Phospholipase D structure and regulation. Chem

Phys Lipids 1999;98:127e40.

Hughes WE, Elgundi Z, Huang P, Frohman MA, Biden TJ. Phospholipase D1

regulates secretagogue-stimulated insulin release in pancreatic beta-cells.

J Biol Chem 2004;279:27534e41.

Jung K, Min DS, Sim KB, Ahn M, Kim H, Cheong J, et al. Upregulation of

phospholipase D1 in the spinal cords of rats with clip compression injury.

Neurosci Lett 2003;336:126e30.

Kim MD, Min DS, Sim KB, Cho HJ, Shin T. Expression and potential role of

phospholipase D1 in cryoinjured cerebral cortex of rats. Histol Histopathol

2004;19:1015e9.

Lauritzen L, Hansen HS. Differential phospholipid-labeling suggests two

subtypes of phospholipase D in rat Leydig cells. Biochem Biophys Res

Commun 1995;217:747e54.

Lee MY, Jo YH, Chun MH, Chung J, Kim M, Min DS. Immunohistochemical

localization of phospholipase D1 in rat central nervous system. Brain Res

2000a;864:52e9.

Lee MY, Kim SY, Min DS, Choi YS, Shin SL, Chun MH, et al. Upregulation

of phospholipase D in astrocytes in response to transient forebrain ische-

mia. Glia 2000b;30:311e7.

Lee EJ, Min DS, Kang WS, Lee MY, Oh SJ, Chun MH. The expression and cellular

localization of phospholipase D1 in rodent retina. Brain Res 2001;905:240e4.

Lee EJ, Min DS, Lee MY, Chung JW, Chun MH, Oh SJ. Differential expres-

sion of phospholipase D1 in the developing retina. Eur J Neurosci 2002;15:

1006e12.

Lee J, Kim H, Ahn M, Min DS, Shin T. Immunohistochemical localization

of phospholipase D1 in the retina of pigs. Neurosci Lett 2006;397:

44e7.

Meier KE, Gibbs TC, Knoepp SM, Ella KM. Expression of phospholipase

D isoforms in mammalian cells. Biochim Biophys Acta 1999;1439:

199e213.

Min DS, Kim EG, Exton JH. Involvement of tyrosine phosphorylation and pro-

tein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3

fibroblasts. J Biol Chem 1998;273:29986e94.

Min DS, Ahn BH, Rhie DJ, Yoon SH, Hahn SJ, Kim MS, et al. Expression and

regulation of phospholipase D during neuronal differentiation of PC12

cells. Neuropharmacology 2001a;41:384e91.

Min DS, Lee KH, Chang JS, Ahn BH, Rhie DJ, Yoon SH, et al. Altered

expression of phospholipase D1 in spontaneously hypertensive rats. Mol

Cells 2001b;11:386e91.

Provost JJ, Fudge J, Israelit S, Siddiqi AR, Exton JH. Tissue-specific distribu-

tion and subcellular distribution of phospholipase D in rat: evidence for

distinct RhoA- and ADP-ribosylation factor (ARF)-regulated isoenzymes.

Biochem J 1996;319:285e91.

Shin T, Min DS, Ahn M, Son W, Matsumoto Y. Increased expression of phos-

pholipase D1 in the sciatic nerve of rats with experimental autoimmune

neuritis. Immunol Invest 2002;31:169e76.

Strand AM, Lauritzen L, Vinggaard AM, Hansen HS. The subcellular locali-

zation of phospholipase D activities in rat Leydig cells. Mol Cell Endocri-

nol 1999;152:99e110.

Vinggaard AM, Hansen HS. Phorbol ester and vasopressin activate phospholi-

pase D in Leydig cells. Mol Cell Endocrinol 1991;79:157e65.

Wetsel WC, Khan WA, Merchenthaler I, Rivera H, Halpern AE, Phung HM,

et al. Tissue and cellular distribution of the extended family of protein

kinase C isoenzymes. J Cell Biol 1992;117:121e33.

Zhao Y, Ehara H, Akao Y, Shamoto M, Nakagawa Y, Banno Y, et al. Increased

activity and intranuclear expression of phospholipase D2 in human renal

cancer. Biochem Biophys Res Commun 2000;278:140e3.