ARTICLE |
Correspondence to:
Robert A. Sikes, U. Virginia Health System, Department of Urology, Molecular Urology and Therapeutics Program, PO Box 800422, Charlottesville, VA 22908-0422. E-mail:
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Summary |
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IGFBP-rP1/mac25 is a recently described member of the insulin-like growth factor binding protein (IGFBP) family. It has structural homology to the other members of the IGFBP family but has a lower affinity for insulin-like growth factors (IGFs). In previous studies using RNA blot hybridization, it was shown that the expression of IGFBP-rP1/mac25 was ubiquitous in normal human tissues. In this report we show by immunohistochemistry that the expression of IGFBP-rP1/mac25 is actually restricted to certain organs and specific cell types. We used an antibody raised against a decapeptide of the C-terminal part of the protein that recognizes a 37-kD protein under reduced conditions. The immunohistochemistry performed on normal human tissues showed a ubiquitous intense staining of peripheral nerves and a variable degree of positive staining in smooth muscle cells, including those from blood vessel walls, gut, bladder, and prostate. Cilia from the respiratory system, epididymis, and fallopian tube showed intense immunoreactivity. Most endothelial cells showed some positivity, whereas fat cells, plasma cells, and lymphocytes were negative. There was specific expression limited to certain cell types in the kidney, adrenal gland, and skeletal muscle, indicating a possible specialized function of IGFBP-rP1/mac25 in these organs. We further noted an opposite pattern of staining in the lining epithelium of breast (typically positive) and prostate glands (largely negative). The specific localization of IGFBP-rP1/mac25 as described implies a function of the protein. However, its regulation within the IGF axis or a possible direct action of IGFBP-rP1/mac25 remains to be demonstrated. (J Histochem Cytochem 48:747754, 2000)
Key Words: growth factors, insulin-like growth factor binding proteins, human, normal expression
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Introduction |
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THE INSULIN-LIKE GROWTH FACTOR (IGF) axis is a complex network of ligands (IGF-I and IGF-II), receptors (IGF-RI and IGF-RII/mannose-6-phosphate receptors) and binding proteins (IGFBP) (
Four groups independently identified proteins that have been determined to be IGFBP-rP1/mac25. One of these groups cloned the mac25 cDNA from normal leptomeningial and mammary epithelial cells; its expression was found to be decreased in the corresponding tumor cells (
Using the above polyclonal antibody, we have analyzed the distribution of IGFBP-rP1/mac25 in normal human tissues to better characterize its cellular localization. Although IGFBP-rP1/mac25 mRNA has been found in almost all tissues by RNA blot hybridization (
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Materials and Methods |
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Cell Lines and Culture
Cell lines were purchased from ATCC (Rockville, MD). Hs578T was cultured in DMEM (GIBCO; Grand Island, NY) supplemented with 10% FBS and 10 µg/ml insulin. MCF-7 cells were cultured in DMEM supplemented with 10% FBS.
Western Blotting
Conditioned media (CM) of MCF-7 and Hs578T were obtained by treating 80% confluent cells in DMEM/F12 without phenol red or serum, and were collected after 48 hr. Ultrafiltration was performed using Centriprep 3 devices (Amicon; Beverly, MA) to obtain an 18.75-fold concentrated CM (concentration followed by desalting in 10 mM Tris, pH 7.4). Protein content was measured by a Bradford colorimetric assay (BioRad; Hercules, CA). Forty µg of protein was loaded under reducing conditions onto a 15% SDS-PAGE gel and transferred to nitrocellulose (MSI; Westborough, MA). The membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20) overnight at 4C. The membranes were rinsed four times in TBST and incubated in a 1:1500 dilution of primary antibody directed against IGFBP-rP1/mac25 diluted in 5% nonfat dry skim milk in TBST for 1 hr at room temperature (RT).
To demonstrate specificity, the rabbit IgG affinity-purified primary antibody (
Immunohistochemistry
Zincformalin-fixed, paraffin-embedded tissues of normal human specimens obtained from surgical pathology samples were sectioned at 5-µm thickness, deparaffinized in xylene, rehydrated in a graded ethanol series, rinsed briefly in PBS, and incubated for 15 min at RT in 10% normal goat serum (Jackson Immunoresearch Laboratories; West Grove, PA). This was followed by treatment with the avidin-biotin blocking kit (Vector; Burlingame CA). After a brief rinse, the sections were incubated overnight at 4C with the T1A12 antibody (1:1500 dilution in 5% normal goat serum, 1% BSA, 0.5% FSG in PBS). After several washes in PBS, the slides were incubated for 30 min at RT with a 1:100 dilution of a biotinylated goat anti-rabbit IgG (Multilink; BioGenex, San Ramon, CA). Endogenous peroxidase was quenched by treating the tissues for 30 min in 0.3% hydrogen peroxide. Slides were then incubated in streptavidinperoxidase diluted 1:60 for 30 min at RT (BioGenex). Peroxidase activity was visualized by a 5-min incubation in diaminobenzidine and hydrogen peroxide (0.01%). The sections were rinsed in water, counterstained with hematoxylin, dehydrated, and mounted.
For the blocking experiments, the primary antibody was first incubated for 1 hr at RT with the peptide (antibody:peptide molar:molar ratio of 1:4). The staining procedure was then performed as described above.
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Results |
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Antibody Characterization
By Western blotting, the polyclonal antibody raised against the decapeptide was able to recognize a single band in Hs578T CM having an apparent Mr of 37 kD under reduced conditions that was not present in MCF-7 CM, in accordance with what was described previously. This band was competed out when the primary antibody was preabsorbed with the decapeptide (molar ratio antibody:peptide of 1:4) (Fig 1C).
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For immunohistochemistry experiments, the primary antibody was preabsorbed following the same procedure with the decapeptide (1:4 ratio) and used to stain a normal breast specimen. The immunoreactive signal observed in epithelium, smooth muscle cells, and nerves (Fig 1A) was lost when the antibody was preabsorbed with the decapeptide (Fig 1B).
IGFBP-rP1/mac25 Immunostaining
All peripheral nerves stained strongly (Fig 2), but the cytoplasm of ganglion cells was variably positive (some cells were moderately positive and others were negative). Some of the supporting cells in peripheral nerves showed nuclear positivity in addition to intense cytoplasmic immunoreactivity. Virtually all smooth muscle cells from assorted sites, such as blood vessel walls, gut, and bladder (Fig 2J), showed moderate positivity, whereas prostate smooth muscle stroma was less intense. Cilia from all sites, including respiratory system, fallopian tube (Fig 2B and Fig 2C), and epididymis (not shown), showed intense staining. Most endothelial cells were positive, with the exception of those in the brain. Fat cells, plasma cells, and lymphocytes (with rare exceptions) lacked staining.
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The results of the positive immunostaining are given in Table 1. Representative examples are shown in Fig 2. Endometrium (Fig 2A) displayed prominent cytoplasmic staining of both proliferative and secretory phase epithelium. The stromal cells were largely negative. In the fallopian tube there was moderate cytoplasmic immunoreactivity of the epithelial lining and of the smooth muscle cells in the wall (Fig 2B). Respiratory bronchial epithelium (Fig 2C) was positive but the alveolar lining cells and pigmented macrophages were negative. In the kidney (Fig 2D), the glomeruli were largely negative, and the epithelia of distal tubules stained stronger than those of proximal tubules. The cells of the adrenal medulla showed some variable reactivity; the cells of the zona reticularis and the zona glomerulosa showed more intense staining than those of the zona fasciculata (Fig 2E). Skeletal muscle (Fig 2F) displayed discrete positivity in some fiber bundles, whereas others were negative. An opposite pattern of immunoreactivity was observed in breast epithelium (Fig 2G) compared to prostate epithelium (Fig 2H). In both organs there was diffuse cytoplasmic staining of smooth muscle cells and strong staining of nerves. The lining epithelium of breast lobules and ducts was immunoreactive, whereas the secretory cells of the prostate were chiefly negative. In the central nervous system, staining was observed in the neuropil with positivity of astrocytes and oligodendrocytes and negativity of neurons (Fig 2I). Immunoreactivity was intense in smooth muscle cells and transitional epithelium of the urinary bladder (Fig 2J). In contrast to the skeletal muscle fiber bundles, there was no mosaic pattern in the bladder smooth muscle tissue. Finally, epithelial cells of the gastrointestinal tract and hepatobiliary system showed weak to moderate immunoreactivity.
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Discussion |
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In this study, we analyzed systematically by immunohistochemistry the distribution of IGFBP-rP1/mac25 in normal human tissue specimens. We used a polyclonal antibody directed against the product of T1A12, a gene identified by a subtractive cDNA cloning strategy using RNAs from the normal breast cell line Hs578Bst and the tumor cell line Hs578T (
Mac25 has been shown to be expressed in all human tissues using a cDNA probe and RNA blot analysis (
Some organs were noted to have compartmentalized staining for IGFBP-rP1/mac25. In the adrenal gland, staining was more intense in the cortex (glomerulosa reticularis > fasciculata) than in the medulla, which displayed variable reactivity.
The staining intensity of IGFBP-rP1/mac25 in the renal tubule epithelium was variable and was consistent with the fact that different parts of the nephron express different IGFBPs (
The staining of some groups of skeletal muscle fibers was positive. whereas others were negative. This could relate to differences in fast- vs slow-twitch muscle fiber bundles, although additional muscle immunohistochemistry would need to be performed. A study performed on rabbit slow and fast skeletal muscle myoblasts showed that there were no constitutive differences in the components of the IGF system between these two fiber types (
In the digestive system, the smooth muscle cells and nerves stained positively. There was some staining of the surface epithelium but not of the mucous glands. There exist only a few studies of IGFBP expression in adult digestive tract. Caco-2 intestinal epithelial cells change their IGFBP secretion profile with their differentiation status (
In addition to the intense staining of cilia, the respiratory bronchial epithelium was uniformly positive. The IGF axis has been believed to play a major role in the development of the rat lung because all IGFBPs show a temporally specific pattern of expression (
In endocrine glands, regulation of the IGF axis by other peptide hormones has been shown. The increase of IGFBP expression in the thyroid gland is accompanied by a decrease in thyroid function (
Intense staining was observed in cilia of fallopian tubes, cervix, endometrium, bronchus, and epididymis. This implies an association of IGFBP-rP1/mac25 with motile structures, because sperm tails also stained intensely. It is possible that IGFBP-rP1/mac25 is critical in motility of cell structures either by regulating the availability of IGFs or by some IGF-independent mechanism.
The data concerning the role of IGFBPs in the female reproductive tract are complex. In the fallopian tube, IGFBP-1 is the predominant binding protein exceeding that of IGFBP-4, -3, and -2 (
In the male genital tract, in contrast to other IGFBPs (-2, -3, -4, and -5) (
In breast and prostate, the involvement of the IGF axis in normal and pathological growth has been demonstrated in several studies (for review see
Perhaps the function of IGFBP-rP is exclusive of IGF. Other members of the IGFBP-rP1 family are induced in response to serum or other growth factors, which places them in the immediate-early response gene category (
The role of IGFBP-rP1 in tumorigenesis is complex. In breast cancer, the loss of IGFBP-rP1 expression was correlated with LOH at chromosome 4q12-13 (
Finally, despite the lack of evidence to date, IGFBP-rP1/mac25 might undergo proteolysis, as shown for IGFBP-3, for which proteolytic fragments can have IGF-independent actions through specific receptors (
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Footnotes |
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1 AD and FW have contributed equally to this work.
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Acknowledgments |
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Supported in part by the Canadian Breast Cancer Research Initiative (AS).
We would like to thank John Sanders and Angelika Burger for fruitful discussions concerning immunohistochemistry. Figure plates were prepared in the University of Virginia Information Technology CenterAcademic Computing Health Sciences.
Received for publication February 9, 2000; accepted February 9, 2000.
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