Cell-cell contact modulates expression of cell adhesion molecule L1 in PC12 cells

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd © 1992 IBRO

Neuroscience Vol. 49, No. 2, pp. 437~,41, 1992 Printed in Great Britain

CELL-CELL CONTACT MODULATES EXPRESSION OF CELL ADHESION MOLECULE L1 IN PC12 CELLS H. KOBAYASHI,* T. MIZUKI, A. WADA a n d F. IZUMI Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Yahatanishi-ku, Kitakyushu, 807 Japan Abstract--The effect of cell contact on the expression of cell adhesion molecule L1 was investigated. The rat pheochromocytoma cell line PC12 cells were cultured at various densities in the presence or absence of the nerve growth factor. The addition of the nerve growth factor promoted the expression of L1. The expression of L1 was higher when the cells were cultured at high density than when done at low density both in the presence or absence of the nerve growth factor. Immunohistochemical staining of L1 showed that the expression of L1 was higher in the cells contacting each other. These results show that cell interaction affects the expression of cell adhesion molecule L1 in the PC12 cells.

Cell to cell c o n t a c t plays a n i m p o r t a n t role in cell differentiation a n d maintenance. 1'4'12 In the past decade, several cell a d h e s i o n molecules which mediate cell contact have been discovered. However, little is k n o w n a b o u t the regulatory m e c h a n i s m s of expression of these molecules. L1 is a cell a d h e s i o n molecule which is involved in n e u r o n - n e u r o n adhesion, neurite fasciculation, outg r o w t h of neurites, cerebellar granule cell m i g r a t i o n a n d neurite o u t g r o w t h on Schwann cells. 5,6,1°AIA3-15 L1 has been s h o w n to be immunologically identical to the nerve growth factor ( N G F ) inducible large external glycoprotein (NILE) which was first identified as a cell-surface glycoprotein in rat PC12 p h e o c h r o m o c y t o m a cell line. 2'9 In this study, to examine the hypothesis t h a t cell~zell contact affects the expression of cell adhesion molecule which in turn m a y m o d u l a t e the cell adhesion, we examined whether adhesion a m o n g cells affects the expression of the L1 in PC12 cells.

EXPERIMENTAL PROCEDURES

Cell culture PC12 cells were kindly provided by Dr J. Meldolesi and maintained in 85% RPMI 1640, 10% horse serum and 5% fetal calf serum. The cells were subcultured on collagen-coated dishes at a density of 5 × 10 3, 1.5 × 104 or 5 × 104 cells/cm2 by adding 2.1, 2.7 or 2 ml of cell suspension (5 × 104 cells/ml) in dishes with an area of 21, 9 or 2 cm 2, respectively, in the presence or absence of 2.5 S NGF (50ng/ml, Collaborative Research, Bedford, MA). When the cell number was counted at the end of

culture, the medium was aspirated and the cells were dissociated by 0.25% trypsin in phosphate-buffered saline (PBS) and counted using a hemocytometer. Immunoblot of L 1 The cells were washed with PBS and collected in a microtube. They were then solubilized by extraction buffer consisting of PBS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1% leupeptin, 100 Kallikrein inhibitor units/ml aprotinin and 0.5% Nonidet P-40, and agitated for 2 h at 4°C. After centrifugation at 20,000 g for 10 min, the resulting supernatant was mixed with a sampling buffer and separated by electrophoresis in 7% polyacrylamide gel containing sodium dodecyl sulfate according to the method of Laemmli. 7 After electrophoresis, proteins were transferred to nitrocellulose filters 16 (0.45 #m) and the L1 was detected by immunoblotting. After pre-incubation with 5% dry milk in PBS, filters were incubated with the IgG fraction of anti-L1 antibody (10#g/ml, polyclonal antibody kindly provided by Dr U. Rutishauser) at 4°C overnight and the bound antibody was reacted with ~25Ilabeled donkey anti-rabbit IgG (1/1,000, Amersham, U.K.) and analysed by a Bioimage Analyser (Fuji Film, Tokyo, Japan). Immunohistochemistry of L 1 The cells were fixed with 4% paraformaldehyde, rinsed with PBS and incubated with 10% goat serum in PBS. L1 was stained by incubation with the IgG fraction of anti-L1 antibody (10 #g/ml) and fluorescein isothiocyanate-labeled anti-rabbit IgG (1/200). The samples were mounted in glycerol-PBS (9:1) containing 2% n-propyl gallate, and observed by fluorescence microscopy.

RESULTS

T o characterize the specificity of o u r a n t i b o d y against L1 a n d to validate quantitativeness in our system, rat b r a i n extract was stained by i m m u n o b l o t . T h e a n t i b o d y recognized 200,000 a n d 140,000 mol. wt b a n d s showing t h a t our a n t i b o d y recognizes L1 specifically, as s h o w n in Fig. 1. F u r t h e r m o r e , the

*To whom correspondence should be addressed. Abbreviations: EDTA, ethylenediaminetetra acetate; NGF, nerve growth factor; NILE, NGF-inducible large external glycoprotein; PBS, phosphate-buffered saline. 437

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expression of L1 was higher when the cells were cultured at high density. The increase in the expression o f L1 by N G F a n d the cell contact was evident from the second day o f culture (Fig. 3). W h e n the cells were cultured for five days, the expression of L1 was increased m u c h more t h a n t h a t cultured for s h o r t periods, a n d the tendency o f increase in L1 expression by cell contact was also seen. These results suggest t h a t the increase in the

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Fig. 1. Immunoblot analysis of L1 of rat brain. Upper panel: rat brain extract was separated by electrophoresis, transferred to nitrocellulose, reacted with anti-L1 antibody and t25I-anti-rabbit IgG. Numbers on the top show the amount (~1) of brain extract (5%) applied. Numbers on the left are the mol. wts x 10 3 of markers: myosin, 200,000 mol. wt; phosphorylase b, 97,000 mol. wt; bovine serum albumin, 69,000 mol. wt; ovalbumin, 46,000 mol. wt. Lower panel: the radioactivity of the 200,000 mol. wt band was quantified from the density of each band by Bioimage Analyser, and the relative intensity was expressed as the ratio to the radioactivity of 10 pl of 5% brain extract.

staining intensity was p r o p o r t i o n a l to the a m o u n t o f b r a i n extract, suggesting o u r system detects L1 quantitatively. L1 antigen was s h o w n to be immunologically identical to the 230,000moi. wt glycoprotein in PC12 cells, which is k n o w n as N I L E . 2 As expected, o u r anti-L1 a n t i b o d y detected a 230,000 mol. wt b a n d in PC12 cells which c o r r e s p o n d s to N I L E (Fig. 2). To see the effect o f cell contact o n L1 expression, PC12 cells were cultured at various cell densities in the presence or absence of N G F for three days. The expression o f L1 increased to m o r e t h a n three-fold by the a d d i t i o n of N G F . B o t h in the presence or absence o f N G F , the

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Fig. 2. Immunoblot analysis of L1 of PC 12 cells cultured for three days. Upper panel: PC12 cells were cultured at 5 × 103 (1, 2), 1.5 × 104 (3, 4) or 5 × 104 (5, 6) cells/cm2 in the presence (2, 4, 6) or absence (1, 3, 5) of NGF for three days. The cells were collected, and L1 was detected as in Fig. 1. Each lane corresponds to 2.5 x 104 cells at the beginning of culture. Lower panel: histogram of L1 expression of PC12 cells. Data are means + S.E.M. of five separate experiments. *P < 0.05, significantly different from the value cultured at low density (1 vs 5; 2 vs 6). Numbers on the bottom show the culture condition as described above. Open bars, without NGF. Shaded bars, with NGF.

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Fig. 3. Time-course of L1 expression in PC12 cells. PC12 cells were cultured at 5 × l0 3 (triangles) or 5 x 104 cells/cm2 (circles) in the presence (closed symbols) or absence (open symbols) of NGF for days indicated in ordinate. The L1 was expressed as in Fig. 1. Each lane corresponds to 2.5 × l 0 4 cells at the beginning of culture. Data are mean _+S.E.M. of five separate experiments.

cell contact during culture increases the expression of L1 in the PC12 cells. To investigate whether the cells proliferate according to cell density, the cell number was counted at the end of culture (Table 1). Three days after plating, the cell number increased to 2.5-2.8-fold irrespective of cell density and the presence of N G F . Five days after plating, the number increased to 7.9-8.8-fold in the absence of N G F and 4.8-5-fold in the presence of N G F , respectively. The cell number did not differ when the cells were cultured at high density or at low density. So the fact that the L1 expression was increased when the cells were cultured at high density rather than at low density indicates that the L1 expression per cell increases by increasing cell contact. To investigate the site of L1 expression of the cell, cells were stained with anti-Ll antibody. N o t only the sites of cell contact were stained but also the cell membrane where it did not come into contact. Both the single cells and contacting cells were stained by anti-L1 antibody, however, the expression of L1 in the contacting cells tends to be stronger than that of single cells. DISCUSSION In this report we have shown that the adhesion among cells regulates the expression of adhesion molecule L1, which in turn may modulate its own adhesiveness of the cells. These regulatory mechanisms of the adhesion molecule may play an important role in the development and maintenance

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of tissues. In our system, the soluble factor secreted from the cells seems not to be involved in the regulation of L1 expression, because the ratio of cell numbers and the amounts of medium were constant when the cells were cultured. Furthermore, in the histochemical staining, the cells that contacted with other cells tended to express much more LI. Cell contact is involved in a variety of mechanisms in the development and maintenance of central and peripheral nervous systems. 1,4A2 N o t only morphological events such as axon guidance, neurite fasciculation and cell migration, but also biochemical and pharmacological characteristics of the cells are also dependent on cell contact. In fact, the activities of neurotransmitter synthesizing enzymes such as tyrosine hydroxylase and choline acetyltransferase are much higher when PC12 cells are cultured at high cell density than those at low density. 8 Furthermore, it is interesting to note that the increase of neurofilament protein response to N G F in PC12 cells was greatly suppressed in co-cultures with the fibroblasts as compared with those with the muscle cells or glioma cells. 3 This suppression was associated with an inhibition of N G F - d e p e n d e n t neurite outgrowth from PC12 cells grown on fibroblast monolayer. Thus, cell-cell interaction can modulate biochemical responses to N G F and it suggests that some kinds of responsiveness of neural cell to environmental factors are dependent on cell-cell interaction. The regulatory mechanism of L1 expression by cell contact has also been present in the neuron with Schwann cell interaction/3 The expression of L1 has been down-regulated by the cell contact between these cells. Thus, the expression of L1 increases or decreases depending on the species of cells or depending on the type of cell contact, that is, contact between the same cells or different cells. Elucidation of the molecular mechanism of the expression of adhesion molecules would provide a clue to clarify the development and maintenance of the nervous system.

Table 1. Cell numbers after culture Cell density (104 cells/cm2) NGF + + +

0 0.5 0.5 1.5 1.5 5.0 5.0

Days after culture 3 1.31 +0.03 1.38 __+0.09 4.19___0.14 4.22 _ 0.23 12.54 _ 0.23 13.51 +0.22

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PC12 cells were cultured at various cell densities in the presence or absence of NGF. At the end of culture, cells were trypsinized and counted. Data are mean __+S.E.M. of four (three-day) or three (five-day) experiments.

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REFERENCES

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