Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor
1997; Springer Nature; Volume: 16; Issue: 1 Linguagem: Inglês
10.1093/emboj/16.1.133
ISSN1460-2075
AutoresSatoshi Ishii, Takahide Nagase, Fumi Tashiro, Koichi Ikuta, Sayuri Sato, Iwao Waga, Kazuhiko Kume, Jun–ichi Miyazaki, Takao Shimizu,
Tópico(s)Immune Cell Function and Interaction
ResumoArticle1 January 1997free access Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor Satoshi Ishii Satoshi Ishii Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Life Science Research Laboratory, Japan Tobacco Inc., Umegaoka 6-2, Aoba-ku, Yokohama, Kanagawa, 227 Japan Search for more papers by this author Takahide Nagase Takahide Nagase Department of Geriatrics, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Fumi Tashiro Fumi Tashiro Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Present address: Institute of Development, Aging and Cancer, Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, Miyagi, 980-77 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Sayuri Sato Sayuri Sato Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Iwao Waga Iwao Waga Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Kazuhiko Kume Kazuhiko Kume Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Jun-ichi Miyazaki Jun-ichi Miyazaki Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Present address: Institute of Development, Aging and Cancer, Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, Miyagi, 980-77 Japan Search for more papers by this author Takao Shimizu Corresponding Author Takao Shimizu Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Satoshi Ishii Satoshi Ishii Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Life Science Research Laboratory, Japan Tobacco Inc., Umegaoka 6-2, Aoba-ku, Yokohama, Kanagawa, 227 Japan Search for more papers by this author Takahide Nagase Takahide Nagase Department of Geriatrics, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Fumi Tashiro Fumi Tashiro Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Present address: Institute of Development, Aging and Cancer, Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, Miyagi, 980-77 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Sayuri Sato Sayuri Sato Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Iwao Waga Iwao Waga Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Kazuhiko Kume Kazuhiko Kume Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Jun-ichi Miyazaki Jun-ichi Miyazaki Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Present address: Institute of Development, Aging and Cancer, Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, Miyagi, 980-77 Japan Search for more papers by this author Takao Shimizu Corresponding Author Takao Shimizu Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Author Information Satoshi Ishii1,2, Takahide Nagase3, Fumi Tashiro4,5, Koichi Ikuta4, Sayuri Sato1, Iwao Waga1, Kazuhiko Kume1, Jun-ichi Miyazaki4,5 and Takao Shimizu 1 1Department of Biochemistry, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan 2Life Science Research Laboratory, Japan Tobacco Inc., Umegaoka 6-2, Aoba-ku, Yokohama, Kanagawa, 227 Japan 3Department of Geriatrics, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan 4Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113 Japan 5Present address: Institute of Development, Aging and Cancer, Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, Miyagi, 980-77 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:133-142https://doi.org/10.1093/emboj/16.1.133 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice overexpressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative abnormalities in the skin were also observed in the transgenic mice, some of which spontaneously bore melanocytic tumors in the dermis after aging. Thus, PAFR transgenic mice proved to be a useful model for studying the basic pathophysiology of bronchial asthma and endotoxin-induced death, and screening of therapeutics for these disorders. Furthermore, our findings provide new insights regarding the role of PAF in the morphogenesis of dermal tissues as well as the mitogenic activity of PAF and PAFR in vivo. Introduction Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator with pleiotropic and potent biological effects on a variety of cells and tissues (Hanahan, 1986; Prescott et al., 1990; Chao and Olson, 1993; Izumi and Shimizu, 1995). Activation of peripheral leukocytes by PAF is thought to be a key step in the pathogenesis of many allergic and inflammatory disorders. Several lines of evidence have also suggested that PAF has various pathophysiological effects on cardiovascular, respiratory and gastrointestinal systems (Stewart and Delbridge, 1993). Moreover, PAF is considered to play physiological roles in the reproductive (O'Neill, 1992; Pike et al., 1992; Toyoshima et al., 1995) and central nervous systems (Bazan, 1990; Kato et al., 1994). The involvement of PAF in these processes has been postulated either by examining effects of exogenously added PAF in vivo or in isolated tissue preparations, by determining the endogenous PAF level, or by examining the modulatory effects of a wide range of PAF antagonists. PAF mediates its biological effects through activation of a G-protein-coupled seven transmembrane receptor (Honda et al., 1991; Chao and Olson, 1993; Izumi and Shimizu, 1995). We and others have cloned PAF receptor (PAFR) cDNAs of guinea-pig (Honda et al., 1991), human (Nakamura et al., 1991; Ye et al., 1991; Kunz et al., 1992; Sugimoto et al., 1992) and rat (Bito et al., 1994) and a PAFR gene of mouse (Ishii et al., 1996). Using these PAFR probes, ubiquitous expression of PAFR mRNA in tissues was demonstrated (Honda et al., 1991; Ye et al., 1991; Bito et al., 1994; Ishii et al., 1996). Notably, leukocytes accumulate enormous amounts of PAFR mRNA. We found two species of human PAFR transcripts, transcript 1 and 2, with identical open reading frames (Mutoh et al., 1993, 1994a, b, 1996). These are driven by individual promoters and are expressed tissue specifically; transcript 1 is ubiquitous and most abundant in leukocytes, while transcript 2 is located in heart, lung, spleen and kidney but not in leukocytes or brain. Such dual promoter systems facilitate the high expression of PAFR mRNA in leukocytes, while maintaining the expression at a lower level in other cells and tissues, under physiological conditions. Augmentation of the message level in other tissues is closely related to the pathogenesis of various disorders; PAFR mRNA significantly increased in the lung tissue of asthmatic patients (Shirasaki et al., 1994). This report is of particular interest since deficiency of plasma PAF acetylhydrolase is associated with severe respiratory symptoms in asthmatic children (Miwa et al., 1988; Stafforini et al., 1996). To elucidate the pathophysiological roles of PAF and PAFR in vivo, we attempted to establish transgenic mice ubiquitously overexpressing PAFR by selecting the β-actin promoter and cytomegalovirus (CMV) enhancer (Niwa et al., 1991). Northern blot analysis demonstrated the expression of the transgene in heart, skeletal muscle, eye, skin, trachea and aorta. PAFR transgenic mice exhibited bronchial hyperreactivity to PAF or methacholine, and an increased lethality to bacterial endotoxin (lipopolysaccharide, LPS). Various unexpected phenotypes included an abnormal breeding pattern, and epidermal and dermal hyperthickening with dermal melanosis. In some aged transgenic mice with a pronounced histopathology in the skin, melanocytic tumors arose spontaneously in the dermis. Thus, these transgenic mice are a pertinent model of bronchial asthma and endotoxin-induced lethality. Furthermore, our findings provide important insights into the novel roles of PAF in cell proliferation and subsequent tumor formation in the skin. Results Establishment of PAFR transgenic mice The guinea-pig PAFR cDNA was placed under the regulation of the chicken β-actin promoter and the CMV immediate early enhancer (Figure 1). A 4.1 kb BamHI-BamHI fragment of the transgene construct (Figure 1) was microinjected into the pronuclei of fertilized eggs. Of 87 offspring, four founders (F0 mice) were identified by PCR screening; at 7-8 weeks old, two of them spontaneously developed necrosis in the hind legs or in the eyes. Though all founders were fertile, only one male founder (F055) transmitted the transgene through the germline. Subsequent analysis of tail DNA of F055 and its offspring transgenic mice by Southern blotting revealed that this line segregated into two sublines based on the copy number of integrated transgenes. This is probably due to two transgene integration sites in the chromosomes of F055 mouse. These two sublines contained ∼50 and 100 copies of the transgene (data not shown), and were designated 55-L and 55-H, respectively. Northern blot analysis of RNAs extracted from heart and skeletal muscle of F2 or F3 mice confirmed the establishment of the two sublines (Figure 2A). When the expression of the transgenic PAFR mRNA was compared in the two groups, 55-H showed 5- to 10-fold higher expression than 55-L. Figure 1.Guinea-pig PAFR transgene expression construct. Open, dotted, hatched and closed boxes represent CMV immediate early (CMV-IE) enhancer, chicken β-actin gene, rabbit β-globin gene and guinea-pig PAFR cDNA, respectively. A polyadenylation [poly(A)] signal is indicated by an arrow head. Splice sites are indicated by arrows. The hybridization probe (a SmaI-SmaI fragment of the guinea-pig PAFR cDNA) used for Southern and Northern blot analysis is also shown. Download figure Download PowerPoint Figure 2.Northern blot analyses. (A) Expression of the PAFR transgene in heart (H) and skeletal muscle (M) of two 55-H and three 55-L transgenic mice. Total RNA (5 μg/lane) was hybridized with the guinea-pig PAFR cDNA probe shown in Figure 1. (B) Expression of the transgene in various tissues of 55-H transgenic mice. Total RNA (3 μg/lane) was hybridized with either the guinea-pig PAFR cDNA probe, the murine PAFR genomic DNA probe or the human GAPDH cDNA probe. Download figure Download PowerPoint Northern blot analysis, using a guinea-pig-specific PAFR probe, of tissue RNAs from 55-H transgenic mice showed a restricted pattern of transgene expression: high levels of expression were seen in heart and skeletal muscle, medium levels in eye, skin, trachea and aorta, and barely detectable levels in neutrophils, brain, lung, liver, spleen, kidney, small intestine, uterus and testis (Figure 2B). Transgenic mRNAs were detected above 18S rRNA. In the control mice, hybridization signals were not observed (data not shown). The membrane was re-hybridized with a murine PAFR probe. Although we have reported the ubiquitous expression of murine PAFR mRNA (Ishii et al., 1996), the murine PAFR mRNA was detected below 28S rRNA only in neutrophils under these conditions because of the low amount of total RNA (3 μg) loaded on the membrane (Figure 2B). The transgene expression pattern of 55-L transgenic mice was similar to that of 55-H (data not shown). Unless stated otherwise, 55-H transgenic mice (F2-F4) were used in the following studies, with their wild-type littermates as control. To verify transgene overexpression in the heart, ligand binding assays were done on cardiac membranes by using a 3H-labeled PAF antagonist, WEB 2086 (Casals-Stenzel, 1987). The membranes were prepared by homogenizing whole hearts from the transgenic and control mice. While membranes of control mice (n = 5) showed no binding activities under our assay conditions, those of the transgenic mice showed high PAFR densities (3.24 ± 0.73 pmol/mg protein, mean ± SEM, n = 4). Anatomical and biochemical examinations No gross morphological abnormalities were detected in three males each of the transgenic mice and their littermate control mice (23 or 35 weeks old) and were found to be normal except for the skin of the ear and tail of the transgenic mice (see below). Light microscopic evaluation of heart, skeletal muscle, thymus, spleen, bone marrow and mesenteric lymph nodes showed no abnormalities. Measurement of body weight of 14- to 17-week-old mice revealed that the transgenic mice were ∼20% lighter than their control littermates of either gender (data not shown). The following parameters were all normal: erythrocyte, platelet and leukocyte counts, proportion and morphology of neutrophils, eosinophils, monocytes and lymphocytes, and serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine phosphokinase, alkaline phosphatase, total cholesterol and creatinine (data not shown). Reduced fertility and gender bias of offspring The transgenic heterozygous females produced rare transgenic progenies: out of 20 pups, only two were transgenic. Thereafter, progeny were generated by mating heterozygous F1 to F3 males to wild-type (BDF1) females. However, the proportion of transgenic progeny to control progeny, 1:2.3 (Table I), was significantly lower than that expected from Mendelian distribution (1:1). In addition, the transgenic ratio among females (1:3.0) was significantly lower than the transgenic ratio among males (1:1.8) (Table I). Each generation of transgenic mice showed similar results. Post-natal deaths were rarely observed. Table 1. Gender and genotype of progeny of 55-H transgenic mice Transgenic Control Female 66a,b 197 Male 103c 186 Total 169 383 The heterozygous transgenic males (F1-F3) of the 55-H line were mated to wild-type (BDF1) females. Data shown are the numbers of progeny of each genotype and gender. P values derived from the χ test: P < 0.001 versus the female control group, P < 0.001 versus the male transgenic group, P < 0.005 versus the male control group. The fertility of 55-L transgenic male was normal with regard to the sex (data not shown) and the transgenic ratio of the progeny (Table II). The mean litter size was 8.2 ± 0.9 (mean ± SEM, n = 10). This value is significantly larger (P < 0.005; two-tailed, unpaired t test) than that observed with the 55-H males that produced progeny under the same conditions [5.5 ± 0.4 (mean ± SEM, n = 24)] (Table II). The reduction of the litter size of the 55-H line roughly corresponded to the decrease of the transgenic ratio. Table 2. Mean litter sizes of PAFR transgenic mice Subline No. in litter No. of progeny Mean litter size ± SEM Transgenic Control Total 55-H 24 32 99 131 5.5 ± 0.4a 55-L 10 40 42 82 8.2 ± 0.9 The heterozygous transgenic males (F1 or F2) of either 55-H or 55-L line were mated to wild-type (BDF1) females. a P< 0.01 versus 55-L group, as determined by two-tailed, unpaired t test. Enhanced sensitivity to PAF First, we assessed the bronchopulmonary effects of PAF by measuring lung resistance in anesthetized, tracheostomized mice. Figure 3A shows typical tracings of tracheal pressure before and after the i.v. administration of 10 μg/kg PAF to the transgenic mouse and to the littermate control mouse. While the control mouse did not respond to PAF, the transgenic mouse showed a marked bronchoconstriction. The response occurred immediately after the administration, peaked in 7 min and continued for >10 min (data not shown). The peak total lung resistance of the transgenic mice was remarkably higher than that of the control mice (2.33 ± 0.40 versus 0.49 ± 0.02 cmH2O/ml/s, mean ± SEM, n = 8 each, P < 0.001; Mann-Whitney U test). Baseline values of the total lung resistance did not differ significantly between transgenic and control mice. A prominent bronchoconstriction and infiltration of blood cells in parenchymal tissues were observed in the transgenic mice (Figure 3B), whereas these changes were not observed in the control mice (Figure 3C). The vehicle of PAF had little or no effect in either group. Figure 3.Bronchopulmonary effects of PAF. (A) Tracheal pressure tracings before and after PAF administration. Mechanically ventilated mice were intravenously administered with PAF (10 μg/kg) and the tracheal pressure changes were monitored continuously. Results shown are arbitrary tracings for 1 s before and after the PAF administration and are representative of the results of eight mice in each group. Typical results of light microscopic analyses of the lungs of (B) PAFR transgenic mouse and (C) control mouse. Note the contracted airway smooth muscle, wrinkled epithelium and blood cell infiltration in alveoli of the transgenic mouse. Hematoxylin and eosin staining. Scale bar (shown in B), 100 μm. Download figure Download PowerPoint Increased bronchopulmonary response to methacholine After nebulized methacholine was administered to mice through the trachea, the total lung resistance was calculated. While high doses of methacholine elicited a substantial bronchoconstriction in the control mice, resultant methacholine dose-response curves (Figure 4A) revealed a significant bronchial hyperreactivity in PAFR transgenic mice. On the basis of the dose-response curves, we evaluated the EC200, the dose required to obtain a 100% increase in the total lung resistance of the baseline. The EC200 of the transgenic mice was significantly smaller than that of the littermate control mice (Figure 4B), suggesting that the airways of transgenic animals were more sensitive to methacholine than those of the controls. The hyperreactivity to methacholine was practically reversed by the prior i.v. administration of a PAF antagonist, WEB 2086 (Figure 4). Even in the control mice, the airway response to methacholine was reduced by the PAF antagonist (Figure 4). Figure 4.Effects of the PAFR transgene and PAF antagonist on reactivity to methacholine. (A) Dose-response curves to methacholine. PAFR transgenic mice or their littermate control mice were administered cumulatively with methacholine aerosols via trachea. The total lung resistance at each dose is represented as RL. WEB 2086 (1.0 mg/kg) was injected i.v. 2 min prior to methacholine. Each group consisted of six mice. Data points represent the mean ± SEM. *P < 0.002 versus each other group, as determined by analysis of variance (ANOVA) with Fisher's PLSD test for pair-wise comparisons. (B) Evaluated EC200 values by the dose-response curves. Statistical significance was determined by ANOVA with Fisher's PLSD test for pair-wise comparisons. Download figure Download PowerPoint Hypersensitivity to endotoxin When we gave Escherichia coli endotoxin (10.0 mg/kg or 12.5 mg/kg i.v.) to the transgenic mice and the littermate control mice, all mice showed clear symptoms of murine endotoxic shock: decreased motor activities, ruffled fur and diarrhea. With a dose of 12.5 mg/kg, the mortality of the transgenic mice was significantly higher (P < 0.02; Fisher's exact test), as compared with that of the control mice (Figure 5): 10% (1/10) of the control mice died within 72 h, whereas 66.7% (6/9) of the transgenic mice died. With a dose of 10.0 mg/kg, there was a trend toward increased sensitivity to the bacterial endotoxin: a mortality of 50% (5/10) for the transgenic mice and 20% (2/10) mortality for the control littermates. Simultaneous injection of WEB 2086 (10 mg/kg) with endotoxin (15 mg/kg) afforded good protection of the transgenic mice from endotoxin-induced death (Table III). Figure 5.Survival curves of mice given bacterial endotoxin. PAFR transgenic females (n = 9) or their littermate control females (n = 10) were injected i.v. with endotoxin (12.5 mg/kg) and survival was monitored for 3 days. *P < 0.02 versus control mice, as determined by Fisher's exact test. Download figure Download PowerPoint Table 3. Protection by WEB 2086 from bacterial endotoxin-induced death of the transgenic mice Endotoxin Endotoxin + WEB 2086a Dead 4 0 Alive 1 4 Survival was monitored for 3 days after the i.v. injection of endotoxin. Numbers of mice are shown. a P < 0.05 versus the endotoxin group, as determined by Fisher's exact test. Hyperplasias of various cutaneous cells and melanocytic tumor formation The transgenic mice had remarkable black patches on the skin of the ear and the tail, irrespective of coat color (Figure 6A); this phenotype was more noticeable in females and was also present in the 55-L transgenic line. Skin pigmentation was appreciable at ∼3-4 weeks old, and increased with age (data not shown). The areas with severe pigmentation were thicker than normal, though the degree of hyperpigmentation differed between individuals. When ear sections of the control mice were stained with hematoxylin and eosin, pigment-containing cells were observed occasionally in the dermis. The epidermis of the control mice consisted of one or two cell layers (Figure 7A). In contrast, the transgenic mice had a large number of pigment-containing cells in the dermis, where hyperplasia of fibroblasts was also observed. In the epidermis, acanthosis (hyperplasia of the epidermis) was evident (Figure 7B). Due to these hyperplasias, the pinna was markedly thickened (compare Figure 7A and B). There were trends for nests of the pigment-containing cells to be present beneath the acanthotic epidermis, and for severe pigmentation to occur at the lateral and the dorsal region of the pinna. To identify the nature of the pigment, these sections were subjected to Fontana-Masson staining, a diagnostic staining for melanin. Since most colored cells were strongly stained (Figure 7C), the pigmentation proved to be due to the increased number of melanin-containing cells. Both hyperthickening of the skin and melanosis in the dermis were also present in the tail (data not shown). In the dermis of the transgenic ear, the number of mast cells increased (data not shown), consistent with a persistent scratching behavior of the transgenic mice. Figure 6.Gross features of PAFR transgenic mice. (A) Ears (upper) and tails (lower) of a 46-week-old transgenic female (left) and a 44-week-old control female (right) with a similar coat color. Note the black patches on the ear and tail of the transgenic mouse. (B) A severely pigmented transgenic mouse (28 months old) with a melanocytic tumor. Several nodules of the tumor are clearly visible. Download figure Download PowerPoint Figure 7.Histopathology of PAFR transgenic mice. (A) The ear section of the control mouse shown in Figure 6A. Hematoxylin and eosin staining. (B) The pigmented skin of the PAFR transgenic ear shown in Figure 6A. Considerable numbers of pigment-containing cells and fibroblasts are present in the dermis. Epidermal hyperthickening (acanthosis) is also observed. Hematoxylin and eosin staining. (C) Fontana-Masson staining of the same section as (B). The pigment-containing cells are mostly positive. Scale bar (shown in A), 50 μm. Download figure Download PowerPoint Electron microscopic examination of the melanin-containing cells in the dermis revealed that they were melanocytes, but not melanophages (Figures 8A and C). The dermal melanocytes of both the control and transgenic mice contained many melanosomes. The majority of them were in mature stage IV (Figures 8B and D). However, in the transgenic mice, melanosomes in developmental stage II and III were frequently observed, indicating the active melanogenesis in PAFR transgenic mice. The melanocytes of transgenic mice were characterized further with enlarged perikarya with well-developed endoplasmic reticula, numerous mitochondria and hypertrophic Golgi complexes (Figure 8D). Figure 8.Electron micrographs of melanocytes in the ear. (A) The dermis of an 11-month-old control female. Scale bar, 5 μm. (B) High-power view of (A). Scale bar, 1 μm. (C) The severely pigmented dermis of a 13-month-old PAFR transgenic female. Scale bar, 5 μm. (D) High-power view of (C). A larger number of premature melanosomes are present than in (B). Melanin contents are low and melanized lamellae are visible. Scale bar, 1 μm. Download figure Download PowerPoint In three out of 14 aged female transgenic mice (>15 months old), melanocytic tumors arose spontaneously in the ear (Figure 6B). One of them also developed a tumor in the tail (data not shown). Histopathological examination revealed that the tumors were located in the dermis (data not shown), suggesting that the tumors derived from the hyperplastic melanocytes. The tumor cells did not metastasize after several months. The control mice showed no such tendency to tumor formation. In summary, multiple abnormalities were observed in the transgenic mice overexpressing PAFR in various tissues, including breeding abnormality, hyperresponsiveness to methacholine or bacterial endotoxin, and aberrant pigmentation and subsequent tumor formation. Discussion Highly deviated expression of the transgene and abnormal breeding Ubiquitous expression of endogenous PAFR mRNA has been reported in all species investigated (Honda et al., 1991; Ye et al., 1991; Bito et al., 1994; Ishii et al., 1996). In an attempt to overexpress PAFR mRNA ubiquitously in transgenic mice, we used the chicken β-actin promoter and CMV enhancer (Niwa et al., 1991), which is known to direct a widespread gene expression (Shimada et al., 1993; Honda et al., 1995). However, the transgene expression was not ubiquitous (Figure 2B). The restricted expression pattern suggests that the ubiquitous overexpression of PAFR may be embryonically lethal to mice. In fact, PAF has been shown to be involved in ovulation, implantation, fetal lung maturation and the initiation and maintenance of parturition (O'Neill, 1992; Pike et al., 1992; Toyoshima et al., 1995). Moreover, failure in the regulation of PAF metabolism has been suggested to elicit premature delivery and infertility (Narahara et al., 1995; Toyoshima et al., 1995). Expression of the PAFR transgene in the early embryonic stage can be deduced from studies of transgenic mice, using the same expression unit (Ikematsu et al., 1993; Araki et al., 1995). A fraction of 55-H transgenic mice, despite the restricted pattern of transgene expression, may not survive gestation (Table II). Thus, even a limited overexpression may still be lethal to some transgenic embryos. The reason for this partial penetrance remains elusive, although the presence of modifying genes that affect the level of the PAFR transgene expression is plausible, as suggested in studies on exencephaly of p53-deficient embryos (Sah et al., 1995). The female transgenic embryos seem to be more difficult to develop than male transgenic embryos (Table I), possibly because the transgene might be expressed more effectively in the female embryos. Alternatively, there might be a sex-dependent effect of PAFR signaling on the embryo. Fertility of the transgenic female is affected by the transgene to a greater extent than that of transgenic males. A higher expression of PAFR in the female reproductive system might easily cause the disorders in fertilization. Enhanced sensitivity to PAF We found abundant expression of PAFR protein in the heart of the transgenic mice. These mice had remarkable bradycardia and arrhythmia immediately after the administration of PAF in doses that elicited a moderate tachycardia in the control mice (unpublished data). The airways of transgenic mice were capable of responding to PAF (Figure 3). Taken together, the overexpressed guinea-pig PAFR pr
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