Journal of Histochemistry and Cytochemistry, Vol. 45, 663-674, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Distribution of Casein-like Proteins in Various Organs of Rat

Makoto Onodaa and Hiroshi Inanoa
a National Institute of Radiological Sciences, Chiba, Japan

Correspondence to: Makoto Onoda, The First Research Group, Natl. Inst. of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba 263, Japan.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Casein-like proteins were detected in various organs of rat by use of a specific antiserum raised against rat milk caseins. The antiserum specifically recognized {alpha}1-, {alpha}2-, ß-, and {gamma}-caseins in rat milk by Western blot analysis, whereas no immunoreactive band was observed in sera of rat and fetal bovine and in bovine caseins. Immunohistochemical studies of this antiserum on formalin-fixed mammary glands showed that immunoreactive caseins were localized to the apical portion of the cytoplasm in lactating mammary epithelial cells and in the luminal secretion, which indicates a directional secretion of caseins to the lumen by the mammary epithelial cells. With this antiserum, immunoreactive substances were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of adrenal gland, distal tubules and convoluted collecting tubules of kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands, and the prickle cell layer of skin, uterine glands and epithelium of the endometrium, hepatic bile ducts, and brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to {alpha}2-casein of rat milk. Skin was the only tissue that expressed both {alpha}2- and ß-caseins. There were no other immunoreactive bands with similarity to ß- and {gamma}-caseins in the other organ extracts, but higher molecular weight immunoreactive bands (> 100 kD) were detected in some organ extracts, such as salivary gland, kidney, liver, lung, and uterus. These findings suggest that the {alpha}2-casein-like substance is localized not only in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs. (J Histochem Cytochem 45:663-674, 1997)

Key Words: casein, antibody, immunohistochemistry, immunoblotting, mammary gland, organ, rat


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Casein is one of the major components of milk, which is produced and secreted by the mammary gland epithelium under hormonal control during lactation (Hobbs et al. 1982 ; Topper and Freeman 1980 ). The caseins are acidic phosphoproteins and constitute almost 80% and >50% of rat (Jenness 1974 ) and mouse (Enami and Nandi 1977 ) milk protein, respectively. Therefore, casein (Hahm et al. 1990 ; Levine and Stockdale 1985 ; Bussolati et al. 1975 ) and {alpha}-lactalbumin (Hall et al. 1979 ; Woods et al. 1979 ) represent specific molecular markers of secretory activity and of the degree of differentiation of mammary gland epithelial cells.

Determination of casein by radioimmunoassay in the serum of patients with various malignancies, not only of the breast but also of other organs, raised concern about the specificity of casein production as a marker of mammary gland origin (Hendrick and Franchimont 1974 ). Pich et al. 1976 demonstrated casein-like proteins immunocytochemically in human tissues, including skin, lung, pancreas, endometrium, and kidney. However, it was reported by Barash et al. 1995 that no immunoreactive caseins were detected by immunoblot analysis in extracts of various tissues from mice, except in the mammary gland. In this context, we have attempted to clarify the presence of caseins and casein-like proteins in a variety of organs, because caseins could be a useful molecular marker to understand the secretory function and the differentiation mechanisms of epithelial cells, not only in mammary gland but in many other organs.

In this article we report the presence and localization of caseins and casein-like proteins in various organs of rat by use of immunohistochemistry and immunoblot analysis with a specific antiserum raised against rat {alpha}-, ß-, and {gamma}-caseins. The possible functional roles of casein-like proteins in organs other than the mammary gland are also discussed.


  Materials and Methods
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Materials and Methods
Results
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Materials and Animals
Reagents and chemicals were purchased from Sigma Chemical (St Louis, MO) unless stated otherwise in the text. All animals used in the present study were treated and handled according to the Recommendations for Handling of Laboratory Animals for Biomedical Research complied by the Committee of the Safety and Handling Regulations for Laboratory Animal Experiments in our Institute.

Preparation of Anti-casein Antiserum
Milk was collected from lactating Wistar-MS rats (Nippon SLC; Hamamatsu, Japan) and partially purified caseins (crude caseins) were obtained from diluted skimmed milk by isoelectric precipitation according to Hahm et al. 1990 . The crude caseins were loaded onto a 12% gel of a minigel system for SDS-PAGE under reducing conditions. The proteins were electrotransferred from the gel to a nitrocellulose membrane (Onoda et al. 1991 ) and visualized with 0.2% Ponceau S. Subsequently, {alpha}-, ß-, and {gamma}-casein bands were excised from the membrane. By using this process the caseins were completely separated from an epithelial membrane antigen (EMA) which was present as an impurity in the crude casein preparations and showed a remarkable similarity of the tissue distribution (Ormerod et al. 1982 ). The casein-bound membranes were incubated together in 250 µl of DMSO for 1 hr to dissolve the membrane. An equal volume of 50 mM sodium carbonate buffer (pH 9.6) was vigorously mixed with the dissolved membrane to obtain a fine particulate suspension of protein/membrane/DMSO (Onoda and Djakiew 1993a , Onoda and Djakiew 1993b ; Abou-Zeid et al. 1987 ). The suspension was emulsified with 500 µl of Freund's complete adjuvant (DIFCO Laboratories; Detroit, MI) and injected SC into a male New Zealand White rabbit (Nippon SLC). Before inoculation, the rabbit was bled for the preparation of pre-immune serum. Six weeks and 12 weeks after the first inoculation, another casein suspension emulsified with Freund's incomplete adjuvant (DIFCO) was injected SC. Three weeks after the last injection the rabbit was bled for the preparation of immune serum. Specificity of the immune serum was confirmed by Western blot analysis of caseins.

Immunoblot Analysis of Rat Caseins
Male and female Wistar-MS rats (8 to 16 weeks old) were sacrificed by decapitation and the following organs were isolated: lactating mammary gland, uterus, pancreas, salivary gland (submandibular gland and sublingual gland), kidney, liver, lung, thymus, brain, adrenal, skin from the abdominal area, testis, and prostate gland. The organs were minced and homogenized in ice-chilled 5 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 5 mM EGTA, and inhibitors (1 M PMSF, 2 mM sodium vanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin) (Onoda and Djakiew 1993a , Onoda and Djakiew 1993b ; van Haren et al. 1992 ). The homogenates were reconstituted in reducing sample buffer and loaded into the minigel system for SDS-PAGE. Subsequently, the separated proteins were electrotransferred to a nitrocellulose membrane (Onoda et al. 1991 ). The membrane was then soaked in 5% non-fat milk in 20 mM Tris-HCl buffer containing 500 mM sodium chloride (TBS, pH 7.5) for 2 hr to block nonspecific immunoreaction, rinsed twice for 10 min in TBS containing 0.05% Tween-20 (TTBS), and reacted overnight with anti-casein antiserum or pre-immune serum (1:5000-20,000 dilution) in TTBS containing 1% gelatin (Bio-Rad Labs; Richmond, CA). The membrane was washed in TTBS twice for 10 min, reacted with either horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000 dilution, Bio-Rad Labs) or alkaline phosphatase-conjugated goat anti-rabbit IgG (1:3000 dilution) in TTBS/gelatin for 1 hr and rinsed in TTBS twice and TBS once. The immunoreactivity was visualized by the following color development reactions (Onoda and Djakiew 1993a , Onoda and Djakiew 1993b ). For the horseradish peroxidase reaction, 4-chloro-1-naphthol (30 mg) was dissolved in ice-cold methanol (10 ml) and hydrogen peroxide (30 µl) was mixed with TBS (50 ml) at room temperature (RT). Both solutions were mixed, and the membrane was incubated in this mixture until color developed. Color development was stopped by replacement of the reaction mixture with distilled water. For the alkaline phosphatase reaction, 5% nitroblue tetrazolium (NBT) in 70% dimethylformamide and 5% 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in dimethylformamide were freshly prepared. NBT and BCIP were then mixed with 100 mM Tris-HCl (pH 9.5) containing 100 mM sodium chloride and 5 mM magnesium chloride (alkaline phosphatase reaction buffer, APB) to a final concentration of 0.033% and 0.017%, respectively. Before color development, the protein-bound nitrocellulose membrane was washed in APB for 10 min and then the immunoreactive bands were visualized in APB containing NBT and BCIP. Color development was terminated by replacement of the reaction mixture with distilled water. The molecular weight of immunoreactive bands was estimated from plots of molecular weight vs relative mobility of rainbow marker standard proteins (Amersham; Arlington Heights, IL) that were run simultaneously with the sample proteins.

Immunohistochemistry of Casein-like Substance in Various Organs
The organs mentioned above were isolated, cut into small cubes, fixed in 10% neutral buffered formalin, usually for about 20 hr, and embedded in paraffin. Paraffin sections (2 µm in thickness) were placed on poly-lysine coated slideglasses, allowed to dry for a few hours on a hotplate at 60C, and left at 37C overnight. The sections were deparaffinized in xylene, rehydrated in descending grades of ethyl alcohol, and brought to PBS. Streptavidin-biotin-peroxidase immunostaining was carried out using Histofine SAB-PO kits (Nichirei; Tokyo, Japan) as described previously (Inano et al. 1995 ). Endogenous peroxidase activity was inactivated by 3% hydrogen peroxide for 15 min at RT, sections were washed twice in PBS for 5 min, then blocked by 10% normal goat serum for 30 min. Next, sections were incubated with anti-casein antiserum or pre-immune serum (1:1000-2000 dilution) at 4C overnight and washed twice in PBS. The sections were treated with biotinylated secondary antibody (goat anti-rabbit IgG) for 30 min at RT, washed twice in PBS, then incubated with streptavidin-peroxidase conjugate for 30 min and washed twice in PBS. Antibody localization on the specimens was visualized by the substrate-chromogen mixture [0.61 M Tris-HCl buffer (pH 7.4) containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide] and color development was stopped by replacement of the reaction mixture with distilled water. The sections were counterstained with hematoxylin, dehydrated, and mounted with a mounting reagent. Photomicrographs were taken under an Olympus BX50 microscope with an automatic camera.


  Results
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Materials and Methods
Results
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Specificity of Anti-casein Antiserum
The crude casein fraction obtained from the milk of Wistar-MS rats contained three major components ({alpha}-, ß-, and {gamma}-caseins), of which the apparent molecular weights were 41.4 ± 0.5 kD, 29.3 ± 0.3 kD, and 24.9 ± 0.3 kD, respectively (Figure 1). These values are similar to those of a previous report (Hahm et al. 1990 ) in which Sprague-Dawley CD rats were used for isolation of milk caseins. Furthermore, these molecular weights were clearly different from that of the EMA, since the EMA did not migrate very much in the running gel (7.5%) of SDS-PAGE and was concentrated at the top of the gel (Ormerod et al. 1982 ). The caseins were transferred to the nitrocellulose membrane and excised to raise a specific antiserum as described in Materials and Methods. The antiserum obtained recognized {alpha}1-, ß-, and {gamma}-caseins in rat milk and crude caseins on the immunoblot analysis even at a dilution ratio of 5000-fold for visualization by the HRP-conjugated secondary antibody (Figure 2). In addition to these three major caseins, an immunoreactive band with an apparent molecular weight of 37.0 kD was detected by the anti-casein antiserum. This molecular species may be {alpha}2-casein, which was immunologically recognized by a specific antibody directed against {alpha}1-casein (Hahm et al. 1990 ; Hennighausen and Sippel 1982 ). Furthermore, the cloned {alpha}-casein cDNA hybridized equally well to both {alpha}-casein-specific mRNAs (Hennighausen and Sippel 1982 ). The {alpha}2-casein was seldom observed in freshly prepared milk (Figure 1) but appeared in milk that had been stored for a longer period at 4C or repeatedly frozen and thawed. Meanwhile, the immunoreactive bands with this antiserum were not detected in rat serum, fetal bovine serum, bovine caseins (Figure 3) nor in mouse serum (data not shown). Furthermore, when the antiserum was absorbed with the crude caseins before immunoblot analysis, the immunoreactive bands were no longer detected by the pre-absorbed antiserum (data not shown). Because these results indicated the specificity of this anti-rat casein antiserum, we decided to employ this antiserum for further immunohistochemical studies of localization of caseins in various organs of rat.



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Figure 1. Partially purified (crude) rat caseins visualized by Coomassie Brilliant Blue staining after SDS-PAGE. Rat milk (10 µg), crude casein (5 µg), and bovine casein (10 µg) were loaded on the minigel system for SDS-PAGE and run as described in Materials and Methods.



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Figure 2. Immunoblot analysis of rat caseins. Immunoreactive caseins in rat milk (Lanes A', 0.25 µg and B', 0.125 µg) and crude rat casein (Lanes C', 0.25 µg and D', 0.125 µg) transferred to nitrocellulose were visualized by Western blot analysis with HRP-conjugated secondary antibody. Another set of milk and crude caseins (Lanes A-D) were not visualized by Coomassie Brilliant Blue (CBB) staining, although the loaded amount of protein was sufficient for visualization by Western blot analysis.



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Figure 3. Immunoblot analysis of various kinds of sera and bovine casein. Two µg of male (M) and female (F) rat sera, fetal bovine serum (FCS), and bovine casein were loaded onto the SDS-PAGE system and the total protein was detected by visualization with CBB. However, the immmunoreactive caseins, in the same set of samples were not recognized by Western blot analysis using the antiserum against rat caseins.

Immunohistochemical Reaction of the Anti-casein Antiserum in Rat Mammary Glands
To validate the effectiveness and usefulness of the anti-casein antiserum for immunohistochemical study regarding the localization of caseins, paraffin-embedded sections were prepared from lactating rat mammary glands and analyzed with the antiserum as described in Materials and Methods. Immunoreactive staining with the antiserum was intense in the apical portion and the supranuclear cytoplasm of the epithelial cells surrounding the lumina of the alveoli and in the secretion of the lumina of the lactating mammary glands (Figure 4B and Figure 4C). This result reconfirms the specificity of the anti-rat casein antiserum, because the anti-EMA antiserum stained only the luminal membranes and the surface of the fat globules, not the cell cytoplasm on lactating breast (Ormerod et al. 1982 ). Similar observations were noted in the primary duct structures of the lactating mammary glands (Figure 4D). In contrast, the immunoreactive signals with the antiserum were barely detected in the apical parts facing the unopened lumina of non-lactating and virgin rat mammary glands (Figure 4E). The absolute amount of reactive staining in virgin rat mammary glands appeared to be minute in comparison with the lactating rat. These findings indicate a directional secretion of caseins to the lumen by the epithelial cells of lactating mammary glands.



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Figure 4. Immunohistochemistry of caseins in rat mammary glands. The mammary glands were isolated from lactating (A-D) and virgin (E) rats and proceeded to immunohistochemistry with a specific anti-rat milk casein antiserum as described in Materials and Methods. (A) A section treated with pre-immune serum. (B-E) Sections treated with anti-casein antiserum. Bars: A, B, E = 50 µm; C = 10 µm; D = 25 µm.

Because the anti-casein antiserum worked on the sections prepared from tissues fixed with 10% neutral buffered formalin and embedded in paraffin, we further investigated the presence and localization of caseins in a variety of organs.

Immunohistochemical Observation of Casein-like Substances in Various Organs
Immunoreactive staining with the anti-casein antiserum was detected in secretory organs such as the pancreas, adrenal gland, and salivary glands (Figure 5). In the pancreas, moderate amounts of casein-like immunoreactive substances appeared in the epithelial cells of pancreatic exocrine ducts and in the endocrine cells of the islets of Langerhans, whereas the immunoreaction in acinar cells was negative (Figure 5A'). In the adrenal gland, cortical cells scattered in the zona fasciculata (Figure 5B') and ganglion cells surrounded by medullary cells were strongly stained with the anti-casein antiserum. Epithelial cells of secretory ducts in both submandibular and sublingual glands showed intensive staining, and some glandular acini that stained brightly with hematoxylin in the submandibular gland were also positive for the antiserum (Figure 5C'). Figure 5 also shows immunoreactive staining with the anti-casein antiserum in the kidney. Intense immunoreaction was detected in the cytoplasm of epithelial cells of distal convoluted tubules, which were distinguished by the lack of brush borders (microvilli) from proximal convoluted tubules (Figure 5D') and collecting tubules. There was no positive staining in proximal tubules and renal corpuscles.



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Figure 5. Immunohistochemical distribution of casein-like proteins in various organs of rat. Sections obtained from pancreas, adrenal gland, salivary gland and kidney were treated with either pre-immune serum (A-D) or anti-casein antiserum (A'-D'). Bars = 100 µm.

Figure 6 shows the casein-like substance recognized with the specific antiserum in liver, lung, skin, and brain. In the liver, hepatic cells surrounding the central vein (Figure 6A') and the cytoplasm of epithelial cells of the hepatic bile duct were apparently positive for the antiserum. In the lung, positive immunoreaction was observed in the epithelium lining the surface of bronchioles and in alveolar epithelium (Figure 6B'). In the skin, the epidermal cell layer (probably the prickle cell layer) was stained strongly, and hair follicles as well as sebaceous glands in the corium layer were intensely stained (Figure 6C'). In the brain, a part of the cerebrum and Ammon's horn was examined for this study. The intensity of immunoreaction with the antiserum was moderate and, in particular, the ventricular epithelium and chorioid plexus in the ventriculus cerebri showed intense immunoreaction (Figure 6D'). Interestingly, a part of the cerebral medulla (perhaps the cingulum bundle/corpus callosum) was relatively intense (Figure 6D').



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Figure 6. Immunohistochemical distribution of casein-like proteins in various organs of rat. Sections obtained from liver, lung, skin and brain (a portion of cerebrum and hippocampus) were treated with either pre-immune serum (A-D) or anti-casein antiserum (A'-D'). Bars = 100 µm.

Figure 7 presents the results of immunoreaction with the anti-casein antiserum in the reproductive and accessory sex organs. In the uterus, the epithelium of the endometrium and the uterine glands were strongly positive to the antiserum (Figure 7A'). However, the immunoreaction to the casein-like substance was inconclusive in the testis and prostate gland (Figure 7B' and 7C'). These results obtained from the immunohistochemical observation of casein-like substances in various organs are summarized with a semiquantitative assessment in Table 1.



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Figure 7. Immunohistochemical distribution of casein-like proteins in reproductive and accessory sex organs of rat. Sections obtained from uterus, prostate, and testis were treated with either pre-immune serum (A-C) or anti-casein antiserum (A'-C'). Bars = 100 µm.


 
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Table 1. Expression of casein-like protein immunoreactivity in rat organs

Determination of Caseins in Various Organ Extracts by Immunoblot Analysis
As expected, the mammary gland extract contained caseins, and {alpha}1-, {alpha}2-, and ß-caseins were abundant in the gland as well as in milk, whereas {gamma}-casein was seldom detectable (Figure 8). The casein-like proteins that immunoreacted with the anti-casein antiserum were detected in many other organ extracts: pancreas, kidney, liver, lung, brain, uterus, skin, adrenal gland, and thymus (Figure 8). In these organ extracts, a major immunoreactive band had approximately a 37-kD molecular weight, which is similar to that of {alpha}2-casein molecular species. In skin extract, a ß-casein-like immunoreactive band was detected, whereas there were no such species of immunoreactive bands in the other organ extracts except for the mammary gland. The salivary gland extract did not contain any casein-like immunoreactive bands, although the secretory ducts of the salivary gland were strongly positive for the anti-casein antiserum in the immunohistochemical study (Figure 5C'). However, the salivary gland contained doublet immunoreactive bands in the higher molecular weight range. Interestingly, similar immunoreactive bands with higher molecular weights appeared in several organ extracts, including the adrenal gland, kidney, liver, lung, skin, uterus, and thymus, and are probably unprocessed precursors of the caseins (Barash et al. 1995 ).



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Figure 8. Immunoblot analysis of caseins and casein-like proteins in homogenates obtained from various organs. The homogenates were loaded onto the SDS-PAGE system and the immunoreactve substances were visualized as described above. The loaded amount of each homogenate was the following: mammary gland (30 µg), pancreas (120 µg), adrenal gland (80 µg), salivary gland (150 µg), kidney (150 µg), liver (150 µg), lung (150 µg), skin (25 µg), brain (150 µg), uterus (100 µg), prostate (80 µg), testis (30 µg), thymus (20 µg) and milk (0.03 µg).


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The ultimate function of the mammary gland obviously is to produce milk, which contains important substances for newborns, and it is known that the growth and differentiation of the mammary gland are basically controlled by multiple interactions of several peptide and steroid hormones from endocrine organs, such as the pituitary and ovary (Topper and Freeman 1980 ). Subsequently, development occurs in several phases, characterized by distinct morphological features. The major element of milk is casein (more than 50% of milk protein) which is synthesized and secreted by the mammary gland epithelium. Therefore, it has been believed that the mammary gland is the sole organ in which caseins are synthesized. Indeed, appreciable amounts of casein mRNAs are present in the rat mammary gland during pregnancy and lactation (Rosen et al. 1975 ), and cDNA clones corresponding to the mRNAs for the lactation-specific proteins, including caseins, have been isolated from a mammary-specific cDNA library (Hennighausen and Sippel 1982 ).

The observation that first localized the casein-like proteins in human organs other than the mammary gland was reported by Pich et al. 1976 . Our present study confirmed the presence of casein-like immunoreactive substances in diverse organs of rat by use of a specific antiserum against rat caseins, although the amounts of casein-like substances in other organs were much less than that in the mammary gland and milk. One noteworthy finding was that the casein-like substances were localized to the epithelium of exocrine ducts in a variety of organs, such as pancreas, salivary gland, kidney, and liver. In addition, the hormonally controlled organs, including the adrenal gland and uterus, also expressed the casein-like substances. Another observation in this study was that the {alpha}2-casein-like protein was a major component of the casein-like protein expressed by organs. To our know-ledge, this is the first time that the {alpha}2-casein molecular species was identified in various organs by Western blot analysis with a specific antiserum. One possible explanation for this phenomenon is the hormonal regulation of casein expression in a variety of organs. It is well understood that the syntheses of the caseins (Terada et al. 1988 ; Enami and Nandi 1977 ; Banerjee 1976 ; Topper 1970 ) and their mRNAs (Hobbs et al. 1982 ; Ono and Oka 1980 ; Guyette et al. 1979 ; Matusik and Rosen 1978 ; Rosen et al. 1978 ; Banerjee 1976 ) are dependent on hormonal control. From these studies, it was deduced that prolactin, hydrocortisone, and insulin were essential for the synthesis of milk proteins and that progesterone suppressed casein synthesis, which is induced and maximized by three essential hormones. In addition, a significant level of mRNAs for caseins was found in virgin rat mammary glands, and the level increased coordinately during pregnancy and lactation (Hobbs et al. 1982 ). The proportions of each of the three casein ({alpha}-, ß-, and {gamma}-) mRNAs remained relatively constant throughout gestation and lactation. The concentration of mRNA for ß-casein became higher than that of other casein mRNAs after gestation, whereas there was more {alpha}-casein mRNA than ß-casein mRNA and the level of {gamma}-casein mRNA was only 2% that of {alpha}-casein mRNA in the virgin rat mammary gland (Hobbs et al. 1982 ). In our study, the {gamma}-casein-like protein was not detected by immunoblot analysis in the various organ extracts obtained from non-pregnant rats, and most of these organs were abundant in the {alpha}-casein-like protein (especially {alpha}2-casein-like protein), except in the salivary gland. Therefore, these observations may reflect the hormonal regulation of casein-like proteins in various organs as well as in the mammary gland.

On the other hand, Ormerod et al. 1982 reported that human casein preparations were associated with small amounts of EMA, which was probably more immunogenic than casein, and that the immunization with such impure casein preparation might yield sera that react with EMA on tissue sections. However, in our present study, {alpha}-, ß-, and {gamma}-casein bands were excised from the nitrocellulose membrane after SDS-PAGE, electrotransfer, and visualization with Ponceau S. Furthermore, the distribution of staining in the lactating mammary glands was different from that obtained with the EMA antiserum, as described in Results. Therefore, it is unlikely that the casein emulsion used for the immunization in our study contained EMA.

Although the localization of caseins in a variety of organs is now certain, the physiological roles of casein and casein-like proteins remain obscure. There are interesting reports that imply some possible roles for caseins. Cytotoxic T-lymphocytes (CTL) expressed members of the casein gene family, such as {alpha}-, ß-, and {kappa}-caseins (Grusby et al. 1990 ). These authors proposed that casein micelles act as a vehicle by which perforin, an important cytolytic mediator released from CTL on antigenic stimulation, is delivered onto the surface of target cells. In that report, the authors also described low levels of mRNA for {alpha}-casein in thymus by Northern blot analysis after PCR amplification. This is consistent with our finding that thymus extract contained an {alpha}2-casein-like substance. Given these observations, caseins may be important as a sort of carrier protein in thymus and in CTL function.

Regarding other aspects, bovine {alpha}-casein was used for construction of a casein-Sepharose affinity column to separate acid proteases from renin in rat brain (Dzau et al. 1982 ; Hirose et al. 1978 ). These studies showed that the acid proteolytic activity, which is similar to that of cathepsin D, bound to the {alpha}-casein-Sepharose, whereas renin in the brain was eluted from the affinity column. Interestingly, the brain acid protease(s) also had angiotensin I-generating activity, which was not inhibited by the anti-renin antibody. These reports state that casein-like immunoreactive substance ({alpha}2-casein) was detected throughout the section of rat brain. Therefore, casein in brain may act as a binding substance for some functional proteins, such as proteases, to regulate their activities and in turn maintain homeostasis. Furthermore, previous demonstrations by Young et al. 1986 and Simon et al. 1987 showed that casein was a substrate for serine esterases from cytolytic T-cells. Therefore, it is not unreasonable that {alpha}-casein may co-exist with such proteases in various organs, and is likely that caseins act as a substrate for certain proteases to control the proteolytic activities of enzymes in a variety of organs.

Although the specific anti-rat casein antiserum we generated provides a useful tool to analyze the mode of expression of the respective genes not only by immunohistochemistry but also by Western blotting, cloned cDNA probes in general would be superior to immunological methods for detection of specific markers in various organs. Therefore, the recognition of mRNAs for caseins or casein-like substances by specific cDNA probes would be a better approach to clarify the presence and localization of casein-like proteins in various organs. Furthermore, certain specific cDNAs will provide better understanding with regard to the regulation of respective genes by steroid and peptide hormones in various organs.


  Acknowledgments

Supported by grants for the Special Project Research of Experimental Studies on the Radiation Health, Detriment and Its Modifying Factors, and for the Research Programme of Bioregulation Mechanism of the National Institute of Radiological Sciences.

We thank Dr Hiroshi Ohtsu, former Director of the Division of Physiology and Pathology, for his review of the immunohistochemical data. We are also grateful to Drs Hiroko Ishii-Ohba and Hiroshi Yamanouchi for their assistance in the preparation of the anti-casein antiserum.

Received for publication August 12, 1996; accepted December 5, 1996.


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

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