Immunohistochemical localisation of the 25 kDa heat shock protein in unstressed ratsPossible functional implications.код для вставкиСкачать
THE ANATOMICAL RECORD 237:453-457 (1993) Immunohistochemical Localisation of the 25 kDa Heat Shock Protein in Unstressed Rats: Possible Functional Implications JENNY M. WILKINSON AND IRINA POLLARD School of Biological Sciences, Macquarie University, Sydney, Australia ABSTRACT The distribution of the 25 kDa heat shock protein (hsp25) in a number of tissue types from unstressed rats was investigated. Immunohistochemical analysis showed that hsp 25 was not found in the thymus, brain (cerebral cortex and cerebellum),testis, adrenal, liver, spleen, or kidney. A number of cells in the anterior pituitary showed strong staining. These cells were tentatively identified as being either gonadotropes or thyrotropes. Strong staining was also observed in the blood vessels within these tissues. Hsp 25 was found to be localised predominantly to intestinal smooth muscle of the duodenum and colon and to vascular smooth muscle. Smooth muscle from other sites, such as the trachea, was also intensely stained. Lower and more variable amounts of staining were observed in cardiac and skeletal muscle. These observations suggest that hsp 25 is associated with cytoskeletal elements in muscle, and that the high staining intensity in smooth muscle might be due to the lack of internal architecture present in this muscle type. o 1993 Wiley-Liss, Inc. Key words: Heat shock proteins, Hsp 25, Immunohistochemistry, Muscle, Rat INTRODUCTION The heat shock proteins (hsps), or stress proteins as they are also known, are a group of highly conserved proteins which are induced by various chemical and mechanical stresses (reviewed in Welch, 1990). The hsps are divided into three general families: hsp 90 (84-90 kDa), hsp 70 (68-75 kDa), and the small hsps (20-30 kDa). The members of these families present in the rat are hsp 90, hsp 71, hsc 73 (heat shock cognate), and hsp 25. Although the hsps are best known for their induction in stressed cells, many of them are expressed in the unstressed cell or have constitutive forms. These proteins, the heat shock cognates (hsc), are only slightly increased during the heat shock response. For example hsp 71 is heat inducible and has low constitutive expression while hsc 73 is constitutively expressed and is only slightly heat inducible. Since hsps are synthesised in unstressed cells it is assumed that they must have an important physiological role in normal cell functioning. The role of the hsp 90 and 70 families has been extensively studied. Hsp 90 has been identified as a component of the steroid hormone receptor complex and the term molecular chaperone has been applied to the group of hsps, including hsp 70, which are found to be an integral part of protein folding, unfolding, and translocation within the cell (Tomasovic, 1989; Jaattela and Wissing, 1992). In contrast to the main groups of mammalian heat shock proteins (hsp 70 and 90) the mammalian small heat shock proteins are poorly characterised in terms of both tissue distribution and physiological function in unstressed cells (Black and Subjeck, 1991). Hsp 25, the small hsp present in rats, is known to exist in several 0 1993 WILEY-LISS, INC. phosphorylated isoforms (Kim et al., 1984; Oesterreich et al., 1990), two of which have been identified as protein kinase C substrates in endothelial cells (Darbon et al., 1990). Hsp 25 has been identified as a major phosphoprotein of intestinal smooth muscle and may be involved in the maintenance of contractions initiated by protein kinase C (Bitar et al., 1991). This protein is believed to be important for the development of thermotolerance (Landry et al., 1989) and accumulates in a differentiation dependent manner in embryonic carcinoma and stem cells (Stahl et al., 1992). This data and the low level constitutive expression reported for many cell types suggests an important role for hsp 25 in the unstressed cell. Analysis of the specific distribution of this protein may help to define its physiological role. To investigate this further we have studied the tissue distribution of hsp 25 in a number of tissue types from unstressed adult rats using both Western blotting and immunohistochemistry . METHODS Male Sprague-Dawley rats, 90-100 days old, were housed three per cage in conditions of constant temperature and humidity (21 ? 0.5"C, 46% relative humidity) and a 12 h:12 h 1ight:dark ratio. Food and water were provided ad libitum. The animals were sacrificed Received March 1, 1993; accepted July 13, 1993. Address reprint requests to Dr. I. Pollard, School of Biological Sciences, Macquarie University, 2109, N.S.W. Australia. 454 J.M. WILKINSON AND I. POLLARD and a range of tissue types removed and processed for either Western blotting or immunohistochemistry. Gel Electrophoresis and Western Blotting Tissues were homogenised in ice-cold PBS (30 mM NaH,PO,, 20 mM NaC1) and the proteins separated using 12.5% one-dimensional SDS-PAGE according to Laemmli (1970). Following electrophoresis the gels were either stained with Coomassie blue or transferred to nitrocellulose (Khyse-Andersen, 1984). Blots were blocked with 3% BLOTTO (skim-milk powder), rinsed with TTBS (20 mM Tris HC1, 500 mM NaC1, 0.05% Tween 20, pH 7.5) and then incubated with a polyclonal mouse anti-hsp 25 antibody (Gaestel et al., 1989) diluted 1:200 with TTBS for 18 h a t room temperature (22°C). Following several washes with TTBS the blot was incubated for 1 h in goat-anti-rabbit IgG conjugated to alkaline phosphatase diluted 1:1,500 in TTBS. Antigen-antibody binding was visualised by the addition of NBTBCIP (nitro blue tetrazoliudbromochlorindoyl phosphate). The lane containing molecular weight markers was removed prior to the blocking step and stained with 0.1% Ponceau S in 5% acetic acid. A kDa 1 2 3 4 5 6 8 5 6 8 94 b 67b 43b 30b 2 0, 1 2 3 4 67b 43b lmrnunohistochemistry Tissues were fixed in Bouin's solution for 6 h at 4"C, washed several times in 70% ethanol, dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin wax. Tissue sections (6 pm) were attached to gelatine coated slides and immunostaining was done using a Vectastain-ABC Kit (Vector Laboratories). All procedures were carried out in a moist chamber at room temperature (22°C). Deparaffinized and hydrated slides were blocked with diluted goat serum (20 min) followed by incubation with anti-hsp 25 antibody (0.2 mg/ml) diluted 1:200 with PBS for 18 h. Slides were then washed with PBS, incubated with biotinylated second antibody (70 min), washed with PBS, and then incubated for 60 min in ABC reagent (avidin:biotinylated horseradish peroxidase complex). Tissue antigen was visualised by the addition of a solution containing hydrogen peroxide (0.02%) and DAB (diaminobenzidine tetrahydrochloride; 1 mg/ml in 0.1 M Tris C1, pH 7.2). Immunostained sections were counterstained with Gill's haematoxylin, cleared, and mounted in DPX. Specificity controls used were (1)omission of primary and/or secondary antibody, (2) substitution of primary antibody with non-immune rabbit serum a t a n equivalent protein concentration, and (3) localisation of a n irrelevant antigen using a polyclonal antibody to rat corticosteroid binding globulin (CBG). The levels of endogenous peroxidase, as determined by incubation of sections with DABhydrogen peroxide only, were negligible in all tissues used. 30 b 20b 14* Fig. 1. Coomassie blue stained gel (A) and Western blot (B) of adult tissues separated by SDS-PAGE. An antibody to murine hsp 25 was used to probe the Western blot. Lanes 1-7 contain 150 pg protein, lane 8 contains 60 pg protein. The position of hsp 25 is indicated by a n arrow. Lane designations are 1:kidney; 2: spleen; 3: lung; 4: heart; 5: testis; 6 brain; 7: liver; 8: skeletal muscle. in the liver. Whole pituitaries also showed a single band of Mr 25,000. lmmunohistochemical Localisation of Hsp 25 Hsp 25 was not observed in the parenchyma of brain (cerebral cortex, Fig. 2A, or cerebellum), thymus (Fig. 2B), kidney, liver, testis (Fig. 2C), adrenal or spleen (Fig. 2D). In the duodenum (Fig. 2E) and colon (Fig. 2F) strong staining was found in both the circular and longitudinal muscle layers as well as the muscularis mucosa and its projections into the villi; no staining was found in the submucosa or epithelium. Other smooth muscle, such as that found in the respiratory tract and Fig. 2. Immunolocalisation of hsp 25 in brain (A), thymus (B), testis (C), spleen (D), duodenum (E),colon (F), skeletal muscle (G), pituitary (H), and aorta (I). Hsp 25 positive staining was located in all vessels (b), vascular smooth muscle (sm), longitudinal and cirWestern blotting of samples of each of the tissues blood cular intestinal smooth muscle (lc), muscularis mucosa (mm) of the used in the immunohistochemistry experiments intestine and in cells of the pituitary (white arrowheads). In skeletal showed that the antibody to hsp 25 detected a single muscle (G) staining was variable with staining ranging from weak band of relative molecular weight (Mr) 25,000 (Fig. 1). (single arrow) to strong (double arrows). No staining was observed in cerebral cortex (cc); thymus medulla (m) or cortex (c); seminiferHighest levels were observed in the brain, spleen, skel- the ous tubules (st);epithelium (e); intestinal villi (v); red pulp (rp)and etal muscle, and the heart. Lower levels were recorded white pulp (wp) of the spleen; connective tissue (ct) or elastin fibres in the testis and kidney with hsp 25 barely detectable (ef). Scale bar = 200 pm. RESULTS Western Blotting Hsp 25 LOCALISATION IN ADULT RATS Fig. 2. 455 456 J.M. WILKINSON AND I. POLLARD trachea, was also positively stained. Skeletal muscle from the abdominal wall (Fig. 2G) and thigh showed variable staining. Staining of the myocardium was significantly weaker and more variable than that of smooth or skeletal muscle. Within the anterior pituitary gland (Fig. 2H) positive staining was observed in the cells lining the sinusoids and also in a small number of cells scattered throughout the gland. These cells were tentatively identified as being either gonadotropes or thyrotropes. In all tissues studied strong staining was recorded in vascular smooth muscle (Fig. 2A-D). This is clearly shown in the aorta (Fig. 21) where staining was observed in the smooth muscle of the artery but not in the elastin fibers. All staining appeared to be cytosolic. Examination of control sections confirmed that the positive staining was due to specific staining with the antibody. similar to that of hsp 25. The function of hsp 25 and aB-crystallin in these sites is unknown. However, it is reported that aB-crystallin and ubiquitin (another small hsp) are increased in migrating chick embryonic cells and in diseases characterised by cytoskeletal reorganisation (Scotting et al., 1991). Since the small hsps show structural homology to cxB-crystallin, perhaps they also have a role in cytoskeletal reorganisation. This may not be an unusual hypothesis since mammalian hsp 70 and 90, and the small hsps of Drosophila have been found in association with actin and vimentin-like intermediate filaments (Koyasu et al., 1986;Leicht et al., 1986; Ohtsuka et al., 1986). Further work is required to determine the precise role of hsp 25 in unstressed cells. DISCUSSION The authors wish to thank Dr. M. Gaestel for providing the antibody to murine hsp 25, Dr. D. Walsh for making the antibody available for our use, S. Ali for providing the antibody to rat CBG, and Jenny Norman and Ron Oldfield for photography. This work was supported by an Australian Research Council Grant to I.P. We have shown that hsp 25 is found in all muscle types with the greatest staining intensity observed in smooth muscle. These results are supported by the recent findings of Kato et al. (1992) who, using an immunoassay, measured the levels of hsp 27 (the human equivalent of murine hsp 25) in various human tissues. The highest levels (> 1ng/mg tissue protein) were observed in intestine, aorta, heart, and skeletal muscle with the lowest levels found in cerebral cortex. These results are well correlated with the presence of either smooth muscle or with the number of blood vessels within the tissue as would be predicted by the results of the present study. The different levels of vascularisation of the various tissues also explains the apparent disparity between the Western blot and immunohistochemistry. The reason for the variation in staining intensity in skeletal muscle is unknown; however, preliminary studies indicate that it may be due to the contractile state of the muscle fibers. We have also located hsp 25 positive cells in the anterior pituitary and have tentatively identified these cells as gonadotropes or thyrotropes. Since Western blotting showed a single band of Mr 25,000 and no cross-reaction with either of these two hormones, this suggests that hsp 25 is present in these cells and that the positive staining was not due to a non-specific reaction with another protein. The tissue distribution of hsp 25 suggests that it has a role in muscular tissue and this suggestion is supported by the few physiological studies done to date. Hsp 27 is found to be involved in the non-calmodulin mediated contractions of intestinal smooth muscle initiated by bombesin or protein kinase C but not those induced by substance P (Bitar et al., 1991). Miron et al. (1991) found that a 25 kDa inhibitor of actin polymerization (25k-IAP) present mainly in smooth cardiac and skeletal muscle had high identity with hsp 27. A direct link between the induction of a specific hsp (hsp 25) and reorganisation of the cytoskeleton was suggested. Interestingly hsp 27 is co-purified from human skeletal muscle with aB-crystallin (Kato et al., 1992). aBcrystallin is a major structural lens protein (Wistow and Piatigorsky, 1988), which is also found in many non-lenticular tissues. The results of the present study show that the non-lenticular distribution of aB-crystallin (Durbin et al., 1991; Bhat and Nagineni, 1988) is ACKNOWLEDGMENTS LITERATURE CITED Bhat, S.P., and C.N. 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