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Immunohistochemical localisation of the 25 kDa heat shock protein in unstressed ratsPossible functional implications.

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THE ANATOMICAL RECORD 237:453-457 (1993)
Immunohistochemical Localisation of the 25 kDa Heat Shock
Protein in Unstressed Rats: Possible Functional Implications
School of Biological Sciences, Macquarie University, Sydney, Australia
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,
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
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 .
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.
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.
94 b
2 0,
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
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.
Western Blotting
Fig. 2.
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.
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
Bhat, S.P., and C.N. Nagineni 1989 a B subunit of lens-specific protein
a-crystallin is present in other ocular and non-ocular tissues. Biochem. Biophys. Res. Commun., 158t319-325.
Bitar, K.H., M.S. Kaminski, N. Hailat, K.B. Cease, and J.R. Strahler
1991HSP27 is a mediator of sustained smooth muscle contraction
in response to bombesin. Biochem. Biophys. Res. Commun., 181:
Black, A.R., and J.R. Subjeck 1991 The biology and physiology of the
heat shock and glucose-regulated stress protein systems. Methods
Achiev. Exp. Pathol. (Basel), 15t126-166.
Darbon, J.M., M. Issandou, J.F. Tourier, and F. Bayard 1990 The
respective 27 kDa and 28 kDa protein kinase C substates in vascular endothelial and MCF-7 cells are most probably heat shock
proteins. Biochem. Biophys. Res. Commun., 168.527-536.
Durbin, E.J., R.P. Erickson, M.L. Van Keuren, R.A. Iacob, and D.M.
Kurnit 1991 Developmental appearance of proteins identified by
two-dimensional gel electrophoresis in mouse gonadal tissue.
Mol. Reprod. Dev., 28t245-248.
Gaestel, M., B. Gross, R. Benndorf, M. Strauss, W. Schunk, R. Kraft,
A. Otto, H. Bohm, J . Stahl, H. Drabsch, and H. Bielka 1989 Molecular cloning, sequencing and expression in Escherichia coli of
the 25-kDa growth-related protein of Ehrlich ascites tumor and
its homology to mammalian stress proteins. Eur. J. Biochem.,
Jaattela, M., and D. Wissing 1992 Emerging role of heat shock proteins in biology and medicine. Ann. Med., 24t249-258.
Kato, K., H. Shinohara, S. Goto, Y. Inaguma, R. Morhisita, and T.
Asano 1992 Copurification of small heat shock protein with a B
crystallin from human skeletal muscle. J . Cell Biol., 267t71187725.
Kim, Y.J., J . Shuman, M. Sette, and A. Przybyla 1984 Nuclear localization and phosphorylation of three 25-kilodalton rat stress proteins. Mol. Cell Biol., 4t468-474.
Koyasu, S., E. Nishida, T. Kadowaki, F. Matsuzaki, K. Iida, F.
Harada, M. Kasuga, H. Sakai, and I. Yahara 1986 Two mammalian heat shock proteins, HSP 90 and HSP100, are actin-binding
proteins. Roc. Natl. Acad. Sci. U.S.A., 8333054-8058.
Kyhse-Andersen, J . 1984 Electroblotting of multiple gels: A simple
amaratus without buffer tank for rauid transfer of Droteins from
pbiyacrylamide to nitrocellulose. J. Biochem. Biopcys. Methods,
Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227t680-685.
Landry, J., P. Chretien, H. Lambed, E. Hickey, and L.A. Weber 1989
Heat shock resistance conferred by expression of the human
HSP27 gene in rodent cells. J . Cell Biol., 109r7-15.
Leicht, B.G., H. Biessmann, K.B. Palter, and J.J. Bonner 1986 Small
heat shock proteins of Drosophila associate with the cytoskeleton.
Proc. Natl. Acad. Sci. U.S.A., 83t90-94.
Miron, T., K. Vancompernolle, J. Vandekerhove, M. Wilchek, and B.
Geiger 1991 A 25-kD inhibitor of actin polymerization is a low
molecular mass heat shock protein. J . Cell Biol., 114r255-261.
Oesterreich, St., R. Benndorf, and H. Bielka 1990 The expression of
the growth-related 25 kDa protein (p25) of Ehrlich ascites tumor
cells is increased by hyperthermic treatment (heat shock).
Biomed. Biochim. Acta, 49:219-226.
Ohtsuka, K., K. Tanabe, H. Nakamura, and C. Sato 1986 Possible
cytoskeletal association of 69,000- and 68,000-dalton heat shock
proteins and structural relations among heat shock proteins in
murine mastocytoma cells. Radiat. Res., 108t34-42.
Scotting, P., H. Mcdermott, and R.J. Mayer 1991 Ubiquitin-protein
conjugates and a B crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early
chicken embryos. FEBS Lett, 285r75-79.
Stahl, J., A.M. Wobus, S. Ihrig, C. Lutsch, and H. Bielka 1992 The
small heat shock protein hsp 25 is accumulated in P19 embryonal
carcinoma cells and embryonic stem cells of line BLC6 during
differentiation. Differentiation 51r33 -37.
Tomasovic, S.P. 1989 Functional aspects of the mammalian heatstress protein response. Life Chem. Rep., 7.33-63.
Welch, W.J. 1990 The mammalian stress response: Cell physiology
and biochemistry of stress proteins. In: Stress Proteins in Biology
and Medicine. R.I. Morimoto, A. Tissieres, and C. Georgopoulos,
eds. Cold Spring Harbor Laboratory Press, New York, pp. 223278.
Wistow, G.J., and J. Piatigorsky 1988 Lens crystallins: The evolution
and expression of proteins for a highly specialized tissue. Annu.
Rev. Biochem.. 57~479-504.
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implications, ratspossible, unstressed, immunohistochemical, heat, protein, shock, localisation, function, kda
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