Production of hyaluronan-dependent pericellular matrix by embryonic rat glial cells

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 88 (1995) 122-125

Short communication

Production of hyaluronan-dependent pericellular matrix by embryonic rat glial cells Katherine A. Deyst l, Bryan P. Toole * Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, USA Accepted 9 May 1995

Abstract

The extracellular matrix of brain is largely composed of aggregates formed by assembly of many proteoglycan and link protein molecules along a hyaluronan polymer backbone. Some cell types construct large, highly hydrated, pericellular matrices or 'coats' from these hyaluronan-mediated aggregates. We show here that embryonic glial cells produce such hyaluronan-dependent pericellular matrices in response to addition of serum or basic fibroblast growth factor plus transforming growth factor-/3. It is proposed that such a matrix is a significant component of the extracellular milieu of the brain, especially during morphogenesis within the developing brain, and that basic fibroblast growth factor and transforming growth factor-/3 regulate its production. Keywords: Hyaluronan; Basic fibroblast growth factor; Transforming growth factor-fl; Extracellular matrix; Embryonic glial cell

Cellular interactions with components of extracellular matrices are crucial to morphogenetic events [9]. The extracellular matrix of brain lacks the characteristic fibrous structure of most other extracellular matrices, and recent studies suggest that it is in large part a network of assemblies of proteoglycans and link proteins along a hyaluronan polymer backbone; these aggregates are similar in many respects to those found in cartilage [4,10,18,20]. Some types of cells, including chondrocytes, produce highly hydrated pericellular matrices or 'coats' that extend 10-20 /zm from the cell surface, and are most easily visualized by their ability to exclude particles [5,8]. These pericellular matrices are structurally tethered to the cell surface and their integrity is dependent on interaction of hyaluronan with proteoglycan and with a cell surface hyaluronan receptor [11-14,16,27]. Such hyaluronan-dependent pericellular matrices have an important influence on tissue morphogenesis during development, especially on cell migration and adhesion [23]. Glial cells produce hyaluronan and retain it at their surface via the hyaluronan receptor, CD44 [1,4]. Thus, in this study, we have investigated

* Fax: (1) (617) 636-0380. 1 Present address: Worcester Foundation for Experimental Biology, 222 Maple Avnue, Shrewsbury, MA 01545 0165-3806/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

whether embryonic rat glial cells assemble a pericellular matrix of the type described above. We show that this is the case and that production of this matrix is induced by serum or a combination of basic fibroblast growth factor (bFGF) and transforming growth factor-/3 (TGF-/3). For preparation of embryonic glial cell cultures, pregnant Sprague-Dawley rats (Charles River Laboratories) were decapitated at embryonic day 14 (El4) and embryos were harvested from the uteri into cold Hanks' balanced salt solution. The embryos were removed from the amnion into cold Hams F-12 medium. Skin and epithelia were teased from cerebral vesicles with sharpened needles and forceps. The vesicles were then transferred to a small amount of fresh Hams F-12. The tissue was disrupted into fine clumps by drawing it up and down through the opening of a syringe. The clumps were then completely dispersed using a 27 gauge needle attached to the syringe. Cells were plated onto poly-D-lysine-coated dishes in growth medium at a density of 2 × 10 4 cells/cm 2. The cells were grown in Dulbecco's modified Eagle medium containing 5% fetal bovine serum, 5% horse serum, Lglutamine, and penicillin/ streptomycin (Gibco, Gaithersburg, MD) at 37°C in a humidified chamber containing 6% CO 2. In order to determine the percentage of glia in the cultures, the cells were fixed, permeabilized and assayed for the cytoskeletal marker, glial fibriUary acidic protein

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[19], using indirect immunofluorescence. Populations which were greater than 95% positive for the marker were used for further experimentation. Pericellular matrices were visualized as zones around the periphery of cells from which particles (formalin-fixed horse red blood cells) are excluded [12]. Subconfluent El4 glial cells were suspended using 0.25% trypsin (Gibco) at 37°C for 5 min. The digestion was stopped using an equal volume of medium containing 0.5% horse serum or 2 units/ml of aprotinin (Sigma, St Louis, MO). Cells were washed once with medium and plated at low density ( 2 - 4 × 105 cells) into 35 mm dishes containing the reagent(s) to be tested. After 16-24 h, the medium was removed and a suspension of red blood cells (1 × 10 ~° cells/ml Ca2+/Mg2+-free phosphate-buffered saline containing 1% bovine serum albumin) was added to the dishes. The red blood cells were allowed to settle for 15 rain and then the culture was observed and photographed using an Olympus CK2 inverted phase-contrast microscope. Production of pericellular matrix was assessed for at least 200 cells for each dish. Cells which showed a phasebright region around their entire periphery, with an average width of greater than or equal to the diameter of one red blood cell, were counted as having a pericellular matrix. Using the particle exclusion assay, we found that 3 0 50% of the embryonic glial cells produce large pericellular matrices in medium containing a high concentration of serum (15% horse serum plus 2.5% fetal bovine serum) (Fig. la). Fewer than 3% of glial cells produce a pericellular matrix at low serum concentrations (0.5% horse serum) (Fig. lc). Hyaluronan hexasaccharides and larger oligomers ( 1 - 2 m g / m l ) , prepared as described previously [3,27], inhibit the formation of glial pericellular matrix in the presence of high serum concentrations (data not shown), thus indicating that the structure of this matrix is dependent on hyaluronan-mediated interactions, as found previously for other cell types exhibiting such matrices [5,1114,16,27].

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Fig. 2. Pericellular matrix production is stimulated by a combination of bFGF and TGF-/3. Increasing concentrations of TGF-fl1 were added to the mediumof the embryonicglial cultures in the presence of 5 ng/ml of bFGF and 0.5% horse serum. Values shown are means of duplicates from 3 experiments(n = 6). Standard error was < 3%, except for bFGF+ 10 ng/ml TGF-/31,where it was 6.5%. Results with TGF-/32 were similarto TGF-/31 (not shown). TGF-/3 and bFGF stimulate the formation of hyaluronan-dependent pericellular matrices by chick embryo limb bud mesodermal cells [17]. Since these regulatory factors are present in developing brain [6,25], we examined their effect on pericellular matrix formation by the glial cells at low serum concentration. Neither TGF-/31 (1-10 ng/ml), TGF-/J 2 (1-10 n g / m l ) nor bFGF (1-100 n g / m l ) alone has a significant effect, but addition of combinations of bFGF and either of the TGF-fls stimulates matrix formation by 20-30% of the cells (Fig. lb; Fig. 2). Nerve growth factor has no effect alone or in combination with bFGF or TGF-fl (data not shown). Neutralizing antibodies to bFGF or TGF-fl partially inhibit pericellular matrix production by the glial cells at high serum concentration (Fig. 3) suggesting that the effect of serum may also be mediated by bFGF and TGF-fl. The antibodies may have inhibited factors present in the serum itself or produced by the glial cells in response to stimulation by other serum factors.

Fig. 1. Visualization of embryonicglial pericellular matricesby particle exclusion. The cells were grown 24 h in high serum (a), low serum supplemented with 5 ng/ml bFGF plus 1 ng/ml TGF-fll (b), or low serum (c). TGF-fl and bFGF were purchased from R&D Systems(Minneapolis, MN). Pericellular matrices are indicated by the large arrowheads. Cells that do not produce a pericellular matrix are partially or fully obscured by particles (small arrows). Bar = 40 ram.

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cells and their pericellular matrix with neighboring neurons would have an integral role in brain development.

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Acknowledgements We thank Dr. Stanley Jacobson for his help with preparation of embryonic glial cells and Drs. Syeda Munaim, Raymond Turner and Shib Banerjee for their help with the assays. This work was supported by NIH Grants DE05838 and HD23681 (B.P.T.).

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References 0 Fig. 3. Neutralizing antibodies to bFGF and TGF-/3 partially inhibit the effect of serum on pericellular matrix production. Antibodies (R&D Systems) to bFGF (40 /xg/ml) and TGF-/3 (20 k~g/ml) were added to high serum-containing medium 30 min before the cells were plated. Cells were grown for 24 h in the medium in the presence or absence of antibodies. Values shown are the means of duplicates (range < 8%) and are normalized to the number of cells showing pericellular matrices grown in the absence of antibody.

The results of this study reinforce previous evidence that glial cells of the developing brain participate in an important manner in directing neuronal architecture. Neurons respond to environmental cues provided by glia in the form of diffusible factors, cell surface signals, and extracellular matrix. One such cue would be production of a hyaluronan-enriched, pericellular matrix around glia, providing hydrated conduits in regions of the brain where neuronal migration or process extension could occur. With respect to this possibility, interaction of hyaluronan with cell surface hyaluronan receptors, such as CD44 and RHAMM, has been shown to promote cell migration in several systems [21-24]. Although the structural integrity of these matrices requires only the interaction of hyaluronan with a proteoglycan and a cell surface hyaluronan receptor [11,13,14], they would be expected to incorporate additional components. For example, in developing brain one of the major proteoglycan components, neurocan, interacts not only with hyaluronan but also with the cell adhesion macromolecules, Ng-CAM and N-CAM, inhibiting their action [7]. Thus the pericellular matrix may also provide a suitable milieu wherein these CAMs and other cell surface macromolecules act together with hyaluronan receptors in modulating cell-cell or cell-matrix interactions of importance to morphogenesis. We have shown here that basic FGF and TGF-/3 stimulate assembly of pericellular matrix by embryonic glial cells. These regulatory factors influence several types of glial behavior [2,15,26], and their effect on the properties of the pericellular matrix is likely to be crucial in eliciting these cellular responses. In turn the dynamic interplay of glial

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