Journal of Histochemistry and Cytochemistry, Vol. 49, 739-748, June 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Extrahepatic Expression of Apolipoprotein A-II in Mouse Tissues: Possible Contribution to Mouse Senile Amyloidosis

Li Fua, Ikuo Matsuyamac, Takuya Chibaa, Yanming Xinga, Tatsumi Korenagaa, Zhanjun Guoa, Xiaoying Fua, Jun Nakayamab, Masayuki Moria, and Keiichi Higuchia
a Department of Aging Angiology, Shinshu University School of Medicine, Matsumoto
b Research Center on Aging and Adaptation and Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto
c Division of Clinical Pathology, Nagano Cancer Detection Center, Matsumoto, Japan

Correspondence to: Keiichi Higuchi, Dept. of Aging Angiology, Research Center on Aging and Adaptation, Shinshu University School of Medicine, Matsumoto 390-8621, Japan. E-mail: khiguchi@sch.md.shinshu-u.ac.jp


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Apolipoprotein A-II (apoA-II), an apolipoprotein in serum high-density lipoprotein, is a precursor of mouse senile amyloid fibrils. The liver has been considered to be the primary site of synthesis. However, we performed nonradioactive in situ hybridization analysis in tissue sections from young and old amyloidogenic (R1.P1-Apoa2C) and amyloid-resistant (SAMR1) mice and revealed that other tissues in addition to the liver synthesize apoA-II. We found a strong hybridization signal in the basal cells of the squamous epithelium and the chief cells of the fundic gland in the stomach, the crypt cells and a small portion of the absorptive epithelial cells in the small intestine, the basal cells of the tongue mucosa, and the basal cells of the epidermis and hair follicles in the skin in both mouse strains. Expression of apoA-II mRNA in those tissues was also examined by RT-PCR analysis. Immunolocalization of apoA-II protein also indicated the cellular localization of apoA-II. ApoA-II transcription was not observed in the heart. Amyloid deposition was observed around the cells expressing apoA-II mRNA in the old R1.P1-Apoa2C mice. These results demonstrate that the apoA-II mRNA is transcribed and translated in various extrahepatic tissues and suggest a possible contribution of apoA-II synthesized in these tissues to amyloid deposition. (J Histochem Cytochem 49:739–747, 2001)

Key Words: amyloidosis, mouse, apoA-II mRNA, in situ hybridization


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ApoA-II is the second most abundant apolipoprotein in high-density lipoprotein (HDL) after apoA-I. The main function of lipoprotein is to transport lipids, but the physiological functions of apoA-II are still unclear. Inactivation of the apoA-II gene in mice suggested a complex role of apoA-II in atherosclerosis, with both anti-atherogenic and pro-atherogenic properties (Weng and Breslow 1996 ). Lipid-free apoA-II mediates the cholesterol efflux from the cell membrane by apolipoprotein–membrane interaction (Yokoyama 1998 ). In addition, apoA-I and apoA-II have been shown to interact with many cellular systems, including spermatozoa, neutrophils, complements, placental tissue, and bacteria and viruses (Handwerger et al. 1987 ; Srinivas et al. 1990 ; Akerlof et al. 1991 ; Blackburn et al. 1991 ; Hamilton et al. 1993 ; Motizuki et al. 1999 ). In mice, apoA-II is a precursor of senile amyloid fibrils (AApoAII), which were first isolated from senescence-accelerated inbred strain (SAMP1) with severe amyloidosis and were later found to be present universally in mice (Higuchi et al. 1986 , Higuchi et al. 1991b ; Yonezu et al. 1986 ). Whereas liver and intestine contribute equally to production of apoA-I and other apolipoproteins, apoA-II has been reported to be mostly synthesized by the liver (Knott et al. 1985 ; Kunisada et al. 1986 ). However, a small amount of apoA-II mRNA has been detected in fetal human intestine by Northern blotting (Hussain and Zannis 1990 ; Le Beyec et al. 1999 ), but none in the adult small intestine (Eggerman et al. 1991 ). To unravel the situation in young and old mice, we performed detailed analysis of apoA-II mRNA expression in specific cell types in extrahepatic tissues using an in situ hybridization technique.

Amyloidosis is defined as a group of biochemically diverse conditions characterized by the extracellular accumulation of fine amyloid fibrils to which normally innocuous soluble proteins polymerize (Sipe 1992 ; Westermark 1998 ). One amino acid substitution, proline to glutamine at position 5 in mouse type C apoA-II protein, markedly accelerates age-associated systemic deposition of AApoAII (Higuchi et al. 1991a ; Naiki et al. 1993 ). Type C apoA-II protein polymerizes to amyloid fibril in vitro, but Type B does not (Naiki et al. 1991 ; Chiba et al. 1999 ). We developed a congenic strain of mice R1.P1-Apoa2C that has the apoA-II gene (Apoa2C) coding amyloidogenic Type C apoA-II of the SAMP1 strains on the genetic background of the SAMR1 strains (Higuchi et al. 1993 , Higuchi et al. 1995 ). Histological studies with SAMP1 and R1.P1-Apoa2C mice indicated that AApoAII is deposited in all tissues except the bone and brain parenchyma in old mice (Takeshita et al. 1982 ; Higuchi et al. 1999 ), but that AApoAII deposition was first observed in the papillary layer of the tongue, mucosa, and the submucosa and lamina propria of the small intestine and stomach (Higuchi et al. 1983 ; Hogenesch et al. 1986 ). In some systemic amyloidosis, amyloid deposition initiates from particular tissues, e.g., the perifollicular region of the spleen in experimental AA amyloidosis (Hoshii et al. 1997 ) or the gastrointestinal tract in TTR transgenic mice (Kohno et al. 1997 ). The fine anatomic structural characteristics of these tissues have been considered responsible for these findings. We hypothesized that apoA-II might be synthesized and polymerize directly to fibrils locally, as has been suggested for AA-amyloid arthropathy in chicken (Ovelgonne et al. 1999 ) In the present study we found that other tissues in addition to the liver synthesize apoA-II. This finding should be important in elucidating both the mechanism of the lipoprotein metabolism and the pathogenesis of amyloidosis.


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Animals
R1.P1-Apoa2C has the amyloidogenic apoA-II gene (Apoa2C) and SAMR1 has the non-amyloidogenic apoA-II gene (Apoa2b). Both strains were raised under conventional conditions at 24 ± 2C. A commercial diet (CE-2; Nihon CLEA, Tokyo, Japan) and tapwater were available ad libitum. Nine young (3-months-old) and three old (16–18-months-old) R1.P1-Apoa2C mice and three young (3-months-old) SAMR1 mice were used. All mice were anesthetized with ethyl ether and sacrificed by dislocation of the cervical vertebra. The liver, tongue, stomach, small intestine, heart, skin, spleen, adrenal glands, testis, brain, and kidneys were removed immediately. Half of each organ was fixed in 4% paraformaldehyde with diethyl pyrocarbonate-treated PBS (pH 7.4) at 4C overnight, embedded in paraffin, and cut into 6-µm sections for ISH or into 4-µm sections for immunohistochemical staining as well as hematoxylin and eosin staining. RNA was isolated immediately for RT-PCR analysis from the other half of the organs.

Preparation of apoA-II RNA Probe
A mouse apoA-II cDNA isolated from a SAMP1 mouse liver cDNA library (Kunisada et al. 1986 ) was used to prepare the apoA-II RNA probe. An apoA-II cDNA containing the whole coding sequence was sub-cloned into the PstI site of pGEM-3Zf(+) vector and the resultant vector was used as a template for construction of the RNA probe. A digoxigenin (DIG)-labeled antisense RNA probe was obtained using a HindIII-cut template and T7 DNA polymerase with a DIG RNA labeling kit (Boehringer Mannheim; Indianapolis, IN). Similarly, a sense probe was prepared for use as the negative control by using an EcoRI-cut template and SP6 RNA polymerase with the same kit.

In Situ Hybridization of apoA-II Transcripts
Tissue sections were subjected to ISH to detect apoA-II mRNA as described previously (Kawakami and Nakayama 1997 ). In each experiment, both antisense and sense apoA-II DIG–RNA probes were routinely used in serial sections processed in each tissue specimen as positive and negative controls, respectively. After the tissue sections were deparaffinized in Hemo-De (Fujisawa Pharmaceutical Industries; Osaka, Japan), hydrated slides were immersed in 0.2 N HCl for 20 min and then digested with 20 mg/ml proteinase K (Amresco; Solon, OH) at 37C for 20 min, followed by postfixation with 4% paraformaldehyde. These slides were washed with 0.2% glycine for 10 min and then acetylated for 15 min in freshly prepared 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). The hydrated slides were then dehydrated with ethanol, defatted with chloroform, and air-dried. After prehybridization with 50% deionized formamide and 2 x SSC (1 x SSC = 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) overnight at 45C, the slides were hybridized with 0.6 µg/ml antisense or sense probe in 50% deionized formamide, 2.5 mM ethylenediaminetetra-acetic acid (EDTA, pH 8.0), 300 mM NaCl, 1 x Denhardt's solution, 10% dextran sulfate, and 1 mg/ml brewer's yeast tRNA at 45C overnight. After hybridization, the slides were washed in 50% deionized formamide and 2 x SSC for 1 hr at 45C with shaking and digested with 10 mg/ml RNase A at 37C for 30 min. After being washed again in 50% deionized formamide and 2 x SSC for 1 hr at 45C, 1 x SSC, and 50% deionized formamide at 45C for 1 hr, the sections were subjected to immunohistochemistry for detection of the hybridized probes using an alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim). The alkaline phosphatase reaction was visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.

Immunohistochemistry for apoA-II
Immunohistochemical staining for apoA-II protein was carried out on serial sections from the same organ by the avidin–biotinylated horseradish peroxidase complex (ABC) method with specific antiserum (1:4000 diluted) against guanidine-denatured AApoAII, which reacts with both serum apoA-II and AApoAII amyloid fibrils (Higuchi et al. 1983 ). In all tissues of old mice, the intensity of the AApoAII deposition was graded 0 to 4 (0, no deposition; 1, slight; 2, moderate; 3, severe; and 4, most severe) in sections stained immunohistochemically. The amyloid index, the average degree of AApoAII deposition in five major organs (heart, liver, spleen, stomach and skin), in three old R1.P1-Apoa2C mice used here was 3.60 ± 0.40 (mean ± SD).

RT-PCR Analysis
Total RNAs were extracted from the liver, tongue, stomach, small intestine, heart, skin, spleen, adrenal gland, testis, brain, and kidney (RNAeasy Mini Kit; Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 µg total RNA of each tissue (First-strand cDNA Synthesis Kit; Amersham Pharmacia Biotech, Piscataway, NJ) and subjected to PCR amplification with Taq DNA polymerase (Promega; Madison, WI). The specific primers for PCR amplification of apoA-II mRNA were apoA-II-3 (5'-GGAGCTTTGGTTAAGAGACAGG-3') and apoA-II-4 (5'-TCACTTAGCCGCAGGAGCCG-3'). The RT-PCR amplification was carried out in a 50-µl reaction mixture containing 200 µM each dNTP, 1 x buffer containing 1.5 mM MgCl2, 0.1 µM each primer, and 1.25 U of Taq DNA polymerase. The cycling parameters for RT-PCR were initial denaturation of 1 minute at 94C followed by 30 cycles of 30 sec at 94C, 30 sec at 55C, and 1 min at 72C. A 5-µl aliquot of the PCR product was subjected to 1% agarose (Takara; Tokyo, Japan) gel electrophoresis.

Western Blotting Analysis
Two-month-old R1.P1-Apoa2C mice were anesthetized with pentobarbital, perfused intracardially with PBS (pH 7.4), and the liver, small intestine, stomach, tongue, skin, and heart were removed. Tissues were sonicated in the lysis buffer containing 20 mM Tris-HCl (pH 7.0), 1 mM EDTA (pH 8.0), 1 mM EGTA, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A on ice and centrifuged at 16,000 x g for 30 min at 4C. Supernatants were collected as cytosol fractions. Cytosol fractions (50 µg protein) were separated by 16.5% SDS-PAGE with a Tris-Tricine buffer system using 8 M urea instead of glycerol (Schagger and von Jagow 1987 ), electrotransferred to PVDF membrane (Bio-Rad; Hercules, CA) and immunodetected by anti-AApoAII antiserum (1:4000 diluted) as described previously (Higuchi et al. 1997 ).

In Vitro Amyloid Fibril Extension Assay
The Type C apoA-II monomer was purified from crude AApoAII as described previously (Naiki et al. 1989 ). HDL particles with Type B and Type C apoA-II were prepared from serum of 3–6-month-old SAMR1 and R1.P1-Apoa2C mouse serum, respectively, by preparative ultracentrifugation as described previously. Type B apoA-II monomer was purified from HDL of SAMR1 (Higuchi et al. 1999 ). AApoAII amyloid fibril extension was assayed by the fluorometric method, using the fluorescent dye thioflavin T (ThT). The reaction mixture was prepared on ice and contained 50 ng/µl of sonicated AApoAII and 0.68 µg/µl apoA-II protein, or 2.03 µg/µl HDL containing 0.7 µg/µl apoA-II in 25 mM phosphate buffer (pH 7.5), 300 mM urea. The reaction mixture (40 µl) was put into Eppendorf tubes and the reaction was initiated at the same time in a heat block set at 37C. At each designated incubation time (0–48 hr), the reaction was stopped by cooling on ice and the preparation was subjected to fluorescence spectroscopy, using a spectrophotometer RF-1500 (Shimadzu; Tokyo, Japan). The assay volume was 1.0 ml with excitation at 450 nm and emission at 482 nm (Naiki et al. 1989 ). The reaction mixture contained 250 nM ThT and 50 mM glycine–NaOH buffer (pH 9.0).


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Distribution of apoA-II mRNA-positive Cells in Tissues
Using the antisense apoA-II probes, apoA-II transcripts were clearly observed in the cytoplasm of positive cells, and a negative control study using the sense probe showed no specific reactivity. In the liver, we found positive hybridization signals in all hepatic parenchymal cells (Fig 1A) in young R1.P1-Apoa2C and SAMR1 mice. However, the intensity of signals for apoA-II was markedly lower in hepatocytes of the old mice (Fig 1B) than in those of the young mice. In general, a strong hybridization signal was observed in specific cells in the stomach, small intestine, tongue, and skin (Table 1) in all mice examined (12 young and three old mice). In the stomach, apoA-II transcripts were restricted to the basal cells of the squamous epithelium and the chief cells of the fundic gland (Fig 1E). In the small intestine, apoA-II transcripts were restricted to the crypt cells and a small portion of the absorptive epithelial cells of intestinal mucosa (Fig 1H). In the tongue, cells with apoA-II transcripts were clearly observed in the basal cells of stratified squamous epithelium (Fig 1K). In the skin, apoA-II transcripts were also noted in the basal cells of the epidermis and hair follicles (Fig 1N). A a weak apoA-II mRNA hybridization signal could be observed in the brain neurons, the lymphocytes in the white pulp of the spleen, and the corona cells of gut-associated lymphoid tissues in the small intestine, in the cortex of adrenal glands, and in the spermatogenic cells, except for spermatozoa in the testis (data not shown). On the other hand, no apoA-II transcripts were seen in the heart (Fig 1Q). The apoA-II-expressing cells and the intensity of the signals in the cells in each tissue were not markedly different among the young R1.P1-Apoa2C, young SAMR1, and old R1.P1-Apoa2C mice, except for the liver (Table 1).



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Figure 1. ISH and IHC for apoA-II synthesis in the tissues of young R1.P1-Apoa2C and SAMR1 mice and old R1.P1-Apoa2C mice. (A) The positive hybridization signals for apoA-II mRNA were observed in the cytoplasm of all hepatocytes in a young mouse (3-month-old R1.P1-Apoa2C). (B) The intensity of signals for apoA-II was markedly decreased in an old mouse (18-month-old R1.P1-Apoa2C). (C) Tiny vesicles of apoA-II protein were observed diffusely in the hepatocytes of a young mouse (3-month-old R1.P1-Apoa2C). Two hepatocytes were intensively stained. (D) In an old mouse, AApoAII was deposited (Grade 4) around the central vein in the liver, extending gradually into the hepatic lobule (18-month-old R1.P1-Apoa2C). (E) ApoA-II transcripts were restricted to the basal cells of squamous epithelium (arrow) and the chief cells of fundic gland (arrowhead) in the stomach of a young mouse (3-month-old R1.P1-Apoa2C). (F) Intracellular immunolocalization of apoA-II in the basal cells and chief cells of a young mouse is indicated by arrow and arrowhead, respectively (3-month-old R1.P1-Apoa2C). (G) AApoAII deposits (Grade 3) in the lamina propria and submucosa in the stomach of an old mouse (16-month-old R1.P1-Apoa2C). (H) ApoA-II mRNA transcripts were restricted to the crypt cells (arrow) and a small portion of the absorptive epithelial cells (arrowhead) of the intestinal mucosa (3-month-old R1.P1-Apoa2C mouse). Counterstaining with hematoxylin. (I) Intracellular immunolocalization of apoA-II in the crypt cell of intestinal mucosa in a young mouse (3-month-old R1.P1-Apoa2C) are indicated by arrow. (J) AApoAII deposits (Grade 3) in the lamina propria and submucosa of the intestinal mucosa in an old mouse (18-month-old R1.P1-Apoa2C). (K) In the tongue, apoA-II transcripts were restricted to the basal cells of stratified squamous epithelium (3-month-old R1.P1-Apoa2C mouse). (L) Immunocytochemical localization of apoA-II demonstrates the presence of apoA-II immunoreactivity with a distribution similar to that of mRNA localization in the tongue of a young mouse (3-month-old R1.P1-Apoa2C). (M) AApoAII deposits (Grade 3) in almost all of the papillae in the tongue of an old mouse (16-month-old R1.P1-Apoa2C mouse). (N) ApoA-II transcripts were restricted to the basal cells of the epidermis and hair follicle (arrows) in the skin of a young mouse (3-month-old R1.P1-Apoa2C). Counterstaining with hematoxylin. (O) Intracellular immunolocalization of apoA-II in the cells expressing apoA-II mRNA of the hair follicle in the skin of a young mouse (3-month-old R1.P1-Apoa2C). (P) AApoAII deposits (Grade 3) in the papillary layer of dermis just beneath the membrane and interstitial tissue around the hair follicle in the skin of an old mouse (16-month-old R1.P1-Apoa2 C). (Q) No apoA-II transcripts were seen in the heart of a young mouse (3-month-old R1.P1-Apoa2C). (R) No apparent evidence of cellular apoA-II immunolocalization in the heart of a young mouse (3-month-old R1.P1-Apoa2C). (S) In an old mouse, extensive AApoAII deposition (Grade 3) was observed in myocardium and capillaries (18-month-old R1.P1-Apoa2C). Bars: A,B = 3 µm; C,E,F,I,K,L,N,O = 6 µm; G,H,J,M,P,S = 12 µm; D,Q,R = 30 µm.


 
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Table 1. Summary of ISH, immunohistochemistry, and RT-PCR for apoA-II

Immunohistochemistry for apoA-II Expression
In the young mice of both strains in which no AApoAII amyloid deposition was observed, the localization of ISH signal was entirely consistent with the pattern of the localization of immunoreactive apoA-II protein in serial sections. However, in the old R1.P1-Apoa2C mice, extensive AApoAII deposition was observed in the extracellular spaces.

In the liver, tiny immunoreactive vesicles appeared diffusely in the cytoplasm in the young mice (Fig 1C). AApoAII was deposited around the central vein, extending gradually into the hepatic lobule (Fig 1D) in the old R1.P1-Apoa2C mice (Table 1). In the stomach, immunoreactive vesicles were weaker in the chief cells than in the basal cells in the young mice (Fig 1F). AApoAII was deposited in the lamina propria and submucosa of the stomach in the old mice (Fig 1G). Similarly in the intestines, weak immunoreactive vesicles were observed in the cells expressing apoA-II mRNA (Fig 1I) in the young mice and AApoAII was deposited in the lamina propria and submucosa (Fig 1J) in the old mice. In the tongue, strong immunoreactive vesicles were seen in the basal cells expressing apoA-II mRNA in the young mice (Fig 1L). AApoAII was deposited in almost all of the papillae of the tongue in the old mice (Fig 1M). In the skin, immunoreactive vesicles were strong in the cells expressing apoA-II mRNA (Fig 1O) and AApoAII was deposited in the papillary layer of dermis just beneath the basement membrane and interstitial tissue around the hair follicles in the skin in the old mice (Fig 1P). Apparent evidence of cellular apoA-II protein immunolocalization was not observed in the heart in the young mice (Fig 1R), but AApoAII deposition was observed in the interstitial tissues between the myocardium and around capillaries in the old mice (Fig 1S). Neither immunoreactive vesicles nor AApoAII deposition was observed in the brain of the young and old mice (data not shown). Although immunoreactive vesicles were not observed in the spleen, testis, and kidney in the young mice (data not shown), extensive AApoAII deposition in these tissues was observed in the old mice (data not shown). In the testis, weakly immunoreactive vesicles were observed in the stromal cells in which apoA-II mRNA was not detected in the young mice (data not shown). In the cytoplasm of adrenal cortex cells, weak immunoreactivity was observed (Table 1).

RT-PCR Amplification
As shown in Fig 2, the RT-PCR products of the expected sizes (320 bp) for apoA-II mRNA were obtained in the liver, stomach, small intestine, tongue, skin, spleen, adrenal gland, testis, brain, and kidney. No RT-PCR products for apoA-II mRNA were obtained in the heart samples.



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Figure 2. RT-PCR analysis for apoA-II mRNA from various tissues of a 3-month-old R1.P1-Apoa2C mouse. The PCR products of the expected sizes (320 bp) for apoA-II mRNA were obtained from all tissues examined except the heart. Lane M, marker ({lambda}/HindIII +{phi}{chi}174/HaeIII); Lane 1, positive control (plasmid pSPA2-16 containing apoA-II cDNA sequence); Lane 2, liver; Lane 3, stomach; Lane 4, small intestine; Lane 5, tongue; Lane 6, skin; Lane 7, heart; Lane 8, spleen; Lane 9, adrenal gland; Lane 10, testis; Lane 11, brain; Lane 12, kidney.

Table 1 summarizes the results of ISH, IHC, and RT-PCR for apoA-II synthesis in the young and old mice.

Detection of apoA-II Protein in Tissues by Western Blotting
Western blotting analysis after Tris–tricine–urea/SDS-PAGE of the cytosol fractions revealed three anti-apoA-II-reactive bands with molecular weights of 6.8, 14, and 30 kD in the cytosol fractions of the liver, small intestine, and stomach, respectively (Fig 3). These molecular weights corresponded to the apoA-II monomeric, dimeric, and tetrameric forms, respectively. In the tongue, only apoA-II monomer was detected. A weak apoA-II monomer was detected in the heart. This small amount of apoA-II might be in the blood, which perfusion could not wash away completely.



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Figure 3. Western blotting analysis for apoA-II protein in various tissues of a 3-month-old R1.P1-Apoa2C mouse. Lane 1, 0.5 µg AApoAII amyloid fibrils. Cytosol fractions were isolated from the liver (Lane 2), small intestine (Lane 3), skin (Lane 4), stomach (Lane 5), tongue (Lane 6), and heart (Lane 7). Monomeric, dimeric and tetrameric apoA-II (arrows) were detected in all tissues examined except for the tongue and heart.

AApoAII Amyloid Fibril Extension with Free apoA-II and HDL In Vitro
When AApoAII amyloid fibrils were incubated with free Type C apoA-II, a rapid increase in fluorescence was observed by 12 h (Fig 4). However, no increase was observed during incubation of AApoAII with HDL isolated from R1.P1-Apoa2C and SAMR1 mice until 48 hr. When AApoAII was incubated with Type B apoA-II, a slight decrease in fluorescence was observed.



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Figure 4. In vitro AApoAII fibril extension kinetics with apoA-II and HDL. Time course of the fluorescence after the initiation of the polymerization reaction. The reaction mixture contained 50 ng/µl of AApoAII fibrils and Type C apoA-II (•), Type B apoA-II ({blacktriangleup}), HDL of R1.P1-Apoa2C ({circ}), and HDL of SAMR1 ({triangleup}) in 25 mM phosphate buffer, pH 7.5, 300 mM urea.


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We successfully demonstrated apoA-II transcripts in several cell types in various tissues fixed with 4% paraformaldehyde and embedded in paraffin. The apoA-II mRNA was transcribed not only in the liver but also in the stomach, small intestine, tongue, and skin. This is a new finding, and we have been careful to exclude other possible reasons for these observations. No hybridization signal was visualized on the serial sections from the same tissue with the sense probe (data not shown). Furthermore, we verified the presence of apoA-II mRNA in the tissues of the same mouse by RT-PCR analysis using apoA-II-specific primers. These results support the conclusion that the positive hybridization signals in the cells were specific for the presence of apoA-II mRNA. In addition, the presence of apoA-II cDNA sequences in the human and mouse database of expressed sequence tags (dbEST: http://www.ncbi.nsh.gov/dEST) of the liver, spleen, lungs, testis, kidney, placenta, gallbladder, activated T-cells, and multiple sclerosis verify the extrahepatic apoA-II mRNA expression. Both apoA-II mRNA and apoA-II immunoreactivities were clearly seen in the hepatocytes in the liver, the basal cells and the chief cells in the stomach, the basal cells of the squamous epithelum in the tongue, the crypt cells in the small intestine, and the basal cells of epidermis and hair follicles in the skin (Table 1). Although the apoA-II immunolocalization was weak in the hepatocytes, the chief cells in the stomach, and the crypt cells in the small intestine, we considered that these cells might secrete apoA-II immediately after synthesis in the cells. These findings revealed that apoA-II was transcribed and synthesized in these extrahepatic tissues.

It remains unclear whether apoA-II proteins synthesized in cells other than hepatocytes have specific roles in cholesterol metabolism and amyloidogenesis. It is noteworthy that the earliest spontaneous AApoAII deposition was observed in the tissues close to the apoA-II-synthesizing cells in the stomach, small intestine, tongue and skin (Higuchi et al. 1998 , Higuchi et al. 1999 ). Nucleation-dependent polymerization is postulated to be a model that explains well the kinetics of fibril formation in amyloidosis (Jarrett and Lansbury 1993 ; Kisilevsky et al. 1995 ; Horwich and Weissman 1997 ). This model consists of two phases, i.e., the nucleation and extension phases. Because the nucleation phase is thermodynamically unfavorable, it is the rate-limiting step in amyloid fibril formation. The mechanism of nucleation has not yet been elucidated, but it should need highly concentrated monomers. Western blotting analysis revealed a considerable amount of apoA-II in the extrahepatic cells. Once the nucleus has been formed, further addition of apoA-II monomer results in rapid extension of amyloid fibrils. The finding that organs with heavy amyloid deposition trapped more radiolabeled apoA-II in reconstituted HDL suggested that circulating apoA-II binds and extends pre-existing amyloid fibrils (Naiki et al. 1988 ). Here, we compared the fibril formation ability of free apoA-II with apoA-II in HDL. Free apoA-II (Type C) extended amyloid fibril rapidly but HDL did not (Fig 4). These results suggest that apoA-II proteins synthesized locally may contribute, at least in part, to nucleation and fibril extension in those tissues. We need further studies to elucidate how much the local tissue synthesis of apoA-II contributies to local amyloid deposition.

Type C apoA-II protein has profound effects on cholesterol metabolism and age-related amyloidosis in mice. The mouse strains that have Type C apoA-II protein have low concentrations of plasma cholesterol and early-onset severe senile amyloidosis. In the liver tissue, the levels of mRNA, translational efficiency, and protein synthesis of apoA-II were no different between mouse strains having Type C and Type B apoA-II (Kitagawa et al. 1994 ; Wang et al. 1997 ). In this experiment, the non-hepatic cells expressing apoA-II mRNA were the same, and we observed no clear difference in the intensity of hybridization and immunohistochemical signals in any cell types of R1.P1-Apoa2C and SAMR1 mice (Table 1). The intensity of positive staining in hepatocytes for apoA-II mRNA was markedly decreased in the old mice compared to the young mice. This is consistent with a previous report (Kitagawa et al. 1994 ), but the mechanism is not yet clear.

In our study, a weak hybridization signal and PCR products for apoA-II mRNA were obtained in the brain, spleen, lung, and testis, but we could not find cellular immunolocalization of apoA-II except in the stromal cells of the testis (Table 1). Because the stromal cells of the testis secrete steroid hormones, the uptake of HDL particles containing apoA-II from the circulation for hormonopoiesis may be responsible for this finding. Additional studies are needed to determine whether apoA-II is synthesized in these tissues. Neither apoA-II mRNA nor immunolocalization was seen in the heart, which suggests that apoA-II is not synthesized in this tissue. AApoAII deposited around the myocardium should come from circulating apoA-II.

In the present study, expression of apoA-II could be detected in the cells with potential proliferation or differentiation activity. The basal cells of squamous epithelium in the stomach, skin, and tongue can proliferate. Hair follicle cells in the skin can also proliferate. The chief cells of the fundic gland can differentiate. In the small intestine, the cells of the bottom of the intestinal crypt are called undifferentiated cells because they move to the top of the intestinal villus with differentiation to the absorptive epithelium. A small part of the absorptive epithelial cells expressed apoA-II mRNA weakly in the present study. This finding is consistent with our previous immunohistochemical findings (Takeshita et al. 1985 ). Disappearance of the transcriptional activity of an apoA-II promoter associated with the differentiation of Caco-2, the human colon adenocarcinoma cell, has been reported (Le Beyec et al. 1999 ). These results, taken together, suggest that the expression of apoA-II may be closely associated with the ability of the cells to proliferate or differentiate.

In conclusion, the present in situ hybridization and immunohistochemical localization studies have demonstrated that apoA-II is transcribed and translated not only in the liver but also in extrahepatic tissues, including the stomach, small intestine, tongue, and skin. These findings may be important for elucidating the apolipoprotein-mediated cholesterol metabolism and pathogenesis of mouse senile amyloidosis. A negative relation between the expression of apoA-II gene and the process of cell differentiation was also suggested.


  Acknowledgments

Supported by Grants-in-Aid on Scientific Research on Priority Areas (09276209) and Scientific Research (B) (114700596) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Ministry of Health and Welfare of Japan.

Received for publication October 26, 2000; accepted January 31, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Akerlof E, Jornvall H, Slotte H, Pousette A (1991) Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility. Biochemistry 30:8986-8990[Medline]

Blackburn WD, Jr, Dohlman JG, Venkatachalapathi YV, Pillion DJ, Koopman WJ, Segrest JP, Anantharamaiah GM (1991) Apolipoprotein A-I decreases neutrophil degranulation and superoxide production. J Lipid Res 32:1911-1918[Abstract]

Chiba T, Kogishi K, Wang J, Xia C, Matsushita T, Miyazaki J, Saito I, Hosokawa M, Higuchi K (1999) Mouse senile amyloid deposition is suppressed by adenovirus-mediated overexpression of amyloid-resistant apolipoprotein A-II. Am J Pathol 155:1319-1326[Abstract/Free Full Text]

Eggerman TL, Hoeg JM, Meng MS, Tombragel A, Bojanovski D, Brewer HB, Jr (1991) Differential tissue-specific expression of human apoA-I and apoA-II. J Lipid Res 32:821-828[Abstract]

Hamilton KK, Zhao J, Sims PJ (1993) Interaction between apolipoproteins A-I and A-II and the membrane attack complex of complement. Affinity of the apoproteins for polymeric C9. J Biol Chem 268:3632-3638[Abstract/Free Full Text]

Handwerger S, Quarfordt S, Barrett J, Harman I (1987) Apolipoproteins AI, AII, and CI stimulate placental lactogen release from human placental tissue. A novel action of high density lipoprotein apolipoproteins. J Clin Invest 79:625-628[Medline]

Higuchi K, Hosokawa M, Takeda T (1999) Senescence-accelerated mouse. Methods Enzymol 309:674-686[Medline]

Higuchi K, Kitado H, Kitagawa K, Kogishi K, Naiki H, Takeda T (1993) Development of congenic strains of mice carrying amyloidogenic apolipoprotein A-II (Apoa2C):Apoa2C reduces the plasma level and the size of high density lipoprotein. FEBS Lett 317:207-210[Medline]

Higuchi K, Kitagawa K, Naiki H, Hanada K, Hosokawa M, Takeda T (1991a) Polymorphism of apolipoprotein A-II (apoA-II) among inbred strains of mice. Biochem J 279:427-433[Medline]

Higuchi K, Kogishi K, Wang J, Xia C, Chiba T, Matsushita T, Hosokawa M (1997) Accumulation of pro-apolipoprotein A-II in mouse senile amyloid fibrils. Biochem J 325:653-659[Medline]

Higuchi K, Matsumura A, Honma A, Takeshita S, Hashimoto K, Hosokawa M, Yasuhira K, Takeda T (1983) Systemic senile amyloid in senescence-accelerated mice: a unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method. Lab Invest 48:231-240[Medline]

Higuchi K, Naiki H, Kitagawa K, Hosokawa M, Takeda T (1991) Mouse senile amyloidosis: ASSAM amyloidosis in mice presents universally as a systemic age-associated amyloidosis. Virchows Arch 60:231-239, b [B]

Higuchi K, Naiki H, Kitagawa K, Kitado H, Kogishi K, Matsushita T, Takeda T (1995) The apoA-II gene and development of amyloidosis and senescence in a congenic strain of mice carrying amyloidogenic apoA-II. Lab Invest 72:75-82[Medline]

Higuchi K, Kogishi K, Wang J, Xia C, Chiba T, Matsuchita T, Hoshii Y, Kawano H, Ishihara T, Yokota T, Hosokawa M (1998) Fibrilization in mouse senile amyloidosis is fibril conformation-dependent. Lab Invest 78:1535-1542[Medline]

Higuchi K, Yonezu T, Kogishi K, Matsumura A, Takeshita S, Kohno A, Matsushita M, Hosokawa M, Takeda T (1986) Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum; apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoprotein. J Biol Chem 261:12834-12840[Abstract/Free Full Text]

Hogenesch H, Gruys E, Higuchi K (1986) Senile amyloidosis. In Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP, Ward JM, eds. Pathobiology of the Aging Mouse. Washington, DC, ILSI Press, 144-237

Horwich AL, Weissman JS (1997) Deadly conformations–protein misfolding in prion disease. Cell 89:499-510[Medline]

Hoshii Y, Kawano H, Cui D, Takeda T, Gondo T, Takahashi M, Kogishi K, Higuchi K, Ishihara T (1997) Amyloid A protein amyloidosis induced in apolipoprotein-E-deficient mice. Am J Pathol 151:911-917[Abstract]

Hussain MM, Zannis VI (1990) Intracellular modification of human apolipoprotein A-II (apoA-II) and sites apoA-II mRNA synthesis: comparison of apoA-II with apoC-II and apoC-III isoproteins. Biochemistry 29:177-187

Jarrett JT, Lansbury PJ (1993) Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73:1055-1058[Medline]

Kawakami M, Nakayama J (1997) Enhanced expression of prostate-specific membrane antigen gene in prostate cancer as revealed by in situ hybridization. Cancer Res 57:2321-2324[Abstract]

Kitagawa K, Naiki H, Takeda T, Higuchi K (1994) Age-associated decreases in the messenger ribonucleic acid level and the rate of synthesis of apolipoprotein A-II in murine senile amyloidsis. Lab Invest 70:565-571[Medline]

Kisilevsky R, Gruys E, Shirahama T (1995) Does amyloid enhancing factor (AEF) exist? Is AEF a single biological entity? Amyloid Int J Exp Clin Invest 2:128-133

Knott TJ, Wallis SC, Robertson ME, Priestley LM, Urdea M, Rall LB, Scott J (1985) The human apolipoprotein A-II gene: structural organization and sites of expression. Nucleic Acids Res 13:6387-6398[Abstract]

Kohno K, Palha JA, Miyakawa K, Saraiva MJ, Ito S, Mabuchi T, Blaner WS, Iijima H, Tsukahara S, Episkopou V, Gottesman ME, Shimada K, Takahashi K, Yamamura K, Maeda S (1997) Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy. Am J Pathol 150:1497-1508[Abstract]

Kunisada T, Higuchi K, Aota S, Takeda T, Yamagishi H (1986) Molecular cloning and nucleotide sequence of cDNA for murine senile amyloid protein: nucleotide substitutions found in apolipoprotein A-II cDNA of senescence accelerated mouse (SAM). Nucleic Acids Res 14:5729-5740[Abstract]

Le Beyec J, Ribeiro A, Schreider C, Chambaz J, Rousset M, Pincon–Raymond M, Cardot P (1999) Illegitimate expression of apolipoprotein A-II in Caco-2 cells is due to chromatin organization. Exp Cell Res 247:373-379[Medline]

Motizuki M, Itoh T, Satoh T, Yokota S, Yamada M, Shimamura S, Samejima T, Tsurugi K (1999) Lipid-binding and antimicrobial properties of synthetic peptides of bovine apolipoprotein A-II. Biochem J 342:215-221[Medline]

Naiki H, Higuchi K, Hosokawa M, Takeda T (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T. Anal Biochem 177:244-249[Medline]

Naiki H, Higuchi K, Nakakuki K, Takeda T (1991) Kinetic analysis of amyloid fibril polymerization in vitro. Lab Invest 65:104-110[Medline]

Naiki H, Higuchi K, Shimada A, Takeda T, Nakakuki K (1993) Genetic analysis of murine senile amyloidosis. Lab Invest 68:332-337[Medline]

Naiki H, Higuchi K, Yonezu T, Hosokawa M, Takeda T (1988) Metabolism of senile amyloid precursor and amyloidogenesis. Age-related acceleration of apolipoprotein A-II clearance in the senescence accelerated mouse. Am J Pathol 130:579-587[Abstract]

Ovelgonne JH, Lamdam WJM, van den Bogaard AEJM, Tooten PCJ, Gielkens ALJ, Peeters BP, Gruys E (1999) Two breeds of chicken with different susceptibility to an amyloidogenic strain of Enterococcus faecalis appear to have identical SAAs. In Kyle RA, Gertz MA, eds. Amyloid and Amyloidosis. New York, Parthenon, 309-392

Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-379[Medline]

Sipe JD (1992) Amyloidosis. Annu Rev Biochem 61:947-975[Medline]

Srinivas RV, Birkedal B, Owens RJ, Anantharamaiah GM, Segrest JP, Compans RW (1990) Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs. Virology 176:48-57[Medline]

Takeshita S, Higuchi K, Hosokawa M, Matsumura A, Higuchi K, Kohno A, Matsushita M, Yonezu T, Takeda T (1985) Morphologic demonstration of cytoplasmic ASSAM-related antigenic substance (CASSAM) by an immunoperoxidase technique. Am J Pathol 121:455-465[Abstract]

Takeshita S, Hosokawa M, Irino M, Higuchi K, Shimizu K, Yasuhira K, Takeda T (1982) Spontaneous age-associated amyloidosis in senescence-accelerated mouse (SAM). Mech Aging Dev 20:13-23[Medline]

Wang J, Kitagawa K, Kitado H, Kogishi K, Matsushita T, Hosokawa M, Higuchi K (1997) Regulation of the metabolism of plasma lipoproteins by apolipoprotein A-II. Biochim Biophys Acta 1345:248-258[Medline]

Weng W, Breslow JL (1996) Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility. Proc Natl Acad Sci USA 93:14788-14794[Abstract/Free Full Text]

Westermark P (1998) The pathogenesis of amyloidosis: understanding general principles. Am J Pathol 152:1125-1127[Medline]

Yokoyama S (1998) Apolipoprotein-mediated cellular cholesterol efflux. Biochim Biophys Acta 1392:1-15[Medline]

Yonezu T, Higuchi K, Tsunasawa S, Takagi S, Sakiyama F, Takeda T (1986) High homology is present in the primary structures between murine senile amyloid protein (ASSAM) and human apolipoprotein A-II. FEBS Lett 203:149-152[Medline]