Tenascin/Hexabrachion in Human Skin: Biochemical Identification and Localization by Light and Electron Microscopy Virginia A
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Tenascin/Hexabrachion in Human Skin: Biochemical Identification and Localization by Light and Electron Microscopy Virginia A. Lightner,*~ Francine Gumkowski,* Darell D. Bigner,§ and Harold P. Erickson* Departments of* Cell Biology, ~;Medicine (Division of Dermatology), and §Pathology, Duke University Medical Center, Durham, North Carolina 27710 Abstract. Tenascin/hexabrachion is a large glycopro- basal layer of sweat gland ducts. The sweat glands tein of the extracellular matrix. Previous reports have themselves did not stain. demonstrated that tenascin is associated with epithe- The distribution of tenascin in the papillary dermis lial-mesenchymal interfaces during embryogenesis and was studied at high resolution by immunoelectron mi- is prominent in the matrix of many tumors. However, croscopy. Staining was concentrated in small amor- the distribution of tenascin is more restricted in adult phous patches scattered amongst the collagen fibers tissues. beneath the basal lamina. These patches were not as- We have found tenascin to be present in normal hu- sociated with cell structures, collagen, or elastic Downloaded from man skin in a distribution distinct from other matrix fibers. proteins. Immunohistochemical studies showed staining Tenascin could be partially extracted from the papil- of the papillary dermis immediately beneath the basal lary dermis by urea, guanidine hydrochloride, or high lamina. Examination of skin that had been split within pH solution. The extracted protein showed a 320-kD the lamina lucida of the basement membrane suggested subunit similar to that purified from fibroblast or a localization of tenascin beneath the lamina lucida. In glioma cell cultures. We have developed a sensitive jcb.rupress.org addition, there was finely localized staining within the ELISA assay that can quantitate tenascin at concentra- walls of blood vessels and in the smooth muscle bun- tions as low as 5 ng/ml. Tests on extracts of the papil- dles of the arrectori pilorem. Very prominent staining lary dermis showed tenascin constituted about was seen around the cuboidal cells that formed the 0.02-0.05 % of the protein extracted. on October 9, 2017 ~NASCIN/hexabrachion is a large oligomeric glyco- Chiquet and Fambrough (8) described the localization of protein of the extracellular matrix. The name hexabra- tenascin in a subclass of dense connective tissue including T chion refers to the unique disulfide bonded six-armed perichondrium/periosteum, ligaments, and tendons as well structure that is the most common form of the glycoprotein as in developing smooth muscle tissues. Vaughan et al., (47) (15, 16). The protein has been independently discovered in also noted the prominence of this protein in cartilage, from a number of laboratories and given several names including: which it could be extracted using high salt. Mackie et al., hexabrachion (15, 16), myotendinous antigen (8, 9), GP250 (35) have reported in vitro studies that suggest tenascin plays protein (6, 7), glial mesenchymal extracellular matrix pro- an active role in the initiation of chondrogenesis. They pro- tein (3, 4), tenascin (2, 10), cytotactin (13, 22), J1 protein (30, posed that tenascin may act by modulating the inhibitory 42), and brachionectin (17, 47; for review, see reference 16). effects of fibronectin on chondrocyte differentiation. Studies characterizing this protein indicate tenascin is a col- Edelman and colleagues (13, 22) have studied the appear- lagenase resistant protein that is distinct from types I, III, IV, ance of tenascin in the developing central nervous system, VI, and VII collagen, fibrillin, laminin, entactin, fibronec- noting specific temporal and spatial patterns of expression. tin, chondronectin, and thrombospondin. They proposed that tenascin, specifically synthesized by glial Several laboratories have described the temporal and spa- cells, plays a role in neuron-glial interactions (22, 24). Other tial localization of tenascin during embryogenesis. This dis- studies suggest tenascin plays a role in migration of neural tribution is much more restricted than that of other ECM crest cells, myelination, and formation of neuromuscular proteins including laminin and fibronectin (1, 2, 8, 10, 13, 17, junctions (12, 40, 42, 44}. 18, 22, 24, 30, 35, 37, 42, 47). Individual laboratories have Tenascin expression during embryonic morphogenesis is focused on different tissues and functional associations, in- also linked to developing epithelial-mesenchymal interfaces. cluding dense connective tissues, developing central nervous Aufderheide et al., (2) reported on the induction of tenascin system, epithelial-mesenchymal interfaces of embryos, and in developing kidney only after the differentiation of an epi- tumor matrix. thelial surface from the underlying mesenchyme. Similar © The Rockefeller University Press, 0021-9525/89/06/2483/11 $2.00 The Journal of Cell Biology, Volume 108, June 1989 2483-2493 2483 findings were reported by this group in developing embryonic anti-tenascin was the same as that seen with 81C6 although, as expected, gut (1). In embryonic skin, several reports have noted promi- the signal was more intense with the polyclonal antibody. Formalin-fixed paraffin-embedded sections were deparaffinized by incu- nent tenascin staining in the mesenchyme beneath develop- bation overnight in xylene followed by serial rehydration through 100%, ing skin appendages, including chicken feather papillae and 95 %, and 80% ethanol. After rinsing with water, the sections were blocked rat hair follicle, tooth bud and mammary gland (8, 10, 13). for 20 min with 10% normal goat serum in PBS. Sections were incubated In contrast to embryonic tissue, tenascin expression is overnight at room temperature in a humidified chamber with afffinity- purified polyclonal antibody at 1 ~tg/ml in blocking buffer, which gave opti- reportedly very restricted in adult tissues. However, promi- mum specific staining. Preimmune control antibody was used at the same nent expression of tenascin in the stroma of several types of concentration. Sections stained with preimmune antibody at 30 ttg/ml gave human tumors was first noted by Bourdon et al., (3, 4). Simi- diffuse background but no specific staining. After washing with PBS, the larly, Chiquet-Ehrismann et al. (10) found the protein ex- bound antibody was visualized by the peroxidase ABC (27) method using pressed in developing embryonic rat mammary tissue but diaminobenzidine (Sigma Chemical Co., St. Louis, MO) as the developer. These sections afforded better preservation of the tissue morphology than absent in normal adult tissue. They observed prominent in- frozen sections. However, the monoclonal antibody 81C6 was not reactive duction of tenascin in the matrix surrounding chemically in- to formalin-fixed sections, so these were stained with the affinity-purified duced mammary carcinomas (10). polyclonal antibody. Several comparisons of these sections with cryosec- Because of the association of tenascin with dense connec- tions stained with monoclonal antibodies showed identical localization. tive tissue, including developing skin, and the matrix sur- rounding some epidermally derived tumors, we have exam- Electron Microscopic Immunolocalization ined adult skin to determine if tenascin expression persists Freshly obtained tissue samples were cut into 2-mm blocks and washed for in adult human skin, to determine its ultrastructural localiza- 4 h at 4°C with PBS. They were then incubated with affinity-purified anti- tion, and to characterize the protein isolated from skin rela- body or preimmune control antibody at 2.5/zg/ml in 20 mM Tris HCI, 0.1% tive to that produced by fibroblast and tumor cells in vitro. BSA, 0.15 M NaCI, 0.1% sodium azide, pH 8.0 (Tris buffer) at 4°C for 43 h. After washing with PBS for 6 h at 4°C, the blocks were incubated with 10- nm gold-conjugated goat anti-rabbit antibody (Janssen Life Sciences Prod- ucts, Piscataway, NJ) diluted 1:2 with Tris buffer at 4°C for 15 h. The blocks Materials and Methods were washed for 6 h at 4°C in PBS and fixed in 2.5% glutaraldehyde in Downloaded from 0.1 M sodium cacodylate for 3 h. The blocks were extensively washed with Antibodies~Antigens 0.1 M sodium cacodylate, stained for 1 h at 4°C in 1% osmium tetroxide in 0.I M sodium cacodylate, washed twice with 0.1 M sodium cacodylate, Monoclonal antibody 81C6, an IgO2b immunoglobulin to human tenascin washed twice with 0.9% NaCI, and stained with 0.5% uranyl acetate in (3, 4), was used for affinity purification of tenascin from culture supernatant 0.9% NaC1 for 2 h at room temperature. After rinsing with 0.9% NaCI, the of a human glioma cell line, U-251 MG clone 3, as previously described blocks were dehydrated through 70%, 95%, and 100% ethanol followed by (18). Tenascin was further purified by gradient sedimentation on a 15-40% propylene oxide. The blocks were embedded in Epon Resin and cut in 60- (vol/vol) glycerol gradient (15, 18) before immunization of rabbits. Antise- nm sections (Ultracut E, Reichert Scientific Instruments, Buffalo, NY). The jcb.rupress.org rum obtained after the second and subsequent boosts was affinity-purified sections were stained for 30 min in 7.5% uranyl acetate, rinsed with water, on a column prepared by coupling 1 nag of the purified tenascin to cyanogen and counterstained 10 rain with Sato lead stain (43). Sections were exam- bromide activated sepharose 2-B (45). The affinity-purified antisera had no ined using an electron microscope (model 300; Philips Electronic Instru- detectable reactivity with collagen types I, III, IV, and V, gelatin, keratin, ments, Inc., Mahwah, N J). laminin, or fibronectin on ELISA (14) and showed no reactivity to fibrillin, collagen types I, III, or VII, laminin, fihronectin, or keratin on Western blotting (46). Antibody from preimmune serum was purified on protein Tissue Extraction on October 9, 2017 A-agarose (Sigma P1406) and used as a negative control in immunolocaliza- Tissue obtained from autopsy or surgical specimens was incubated for tion and immunoblotting. A mouse myeloma IgG2b immunoglobulin of no 72-96 h at 4°C in Scaletta buffer.