Induction of tissue plasminogen activator in differentiated NG108-15 cells

Induction of tissue plasminogen activator in differentiated NG108-15 cells

Int. J. Devl Neuroscience 18 (2000) 145±150 www.elsevier.com/locate/ijdevneu Induction of tissue plasminogen activator in di€erentiated NG108-15 cel...

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Int. J. Devl Neuroscience 18 (2000) 145±150

www.elsevier.com/locate/ijdevneu

Induction of tissue plasminogen activator in di€erentiated NG108-15 cells p Glenn C. Friedman, Nicholas W. Seeds* Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 30 April 1999; received in revised form 17 June 1999; accepted 8 July 1999

Abstract Plasminogen activators may facilitate neurite outgrowth and neuronal migration in the developing nervous system. The expression of tissue plasminogen activator by NG108-15 neuroblastoma grown under a variety of conditions has been explored. High tissue plasminogen mRNA expression correlates with growth conditions which induce morphological di€erentiation and neurite outgrowth; however, NG108-15 cells grown in suspension with dibutyryl-cAMP also show a high level of tissue plasminogen activator expression. 7 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Tissue plasminogen activator; Neuroblastoma di€erentiation; mRNA induction

1. Introduction The plasminogen activators (PAs), tissue-type (tPA) or urokinase-type (uPA), are serine proteases that convert the inactive zymogen plasminogen into the active protease plasmin, which is able to degrade many cellular proteins, while activating others such as procollagenase [3,8]. PAs have been implicated in a number of cellular processes including cell migration, tissue activation, and tissue remodeling [3]. PAs are synthesized by a variety of neuronal cell types including cerebellar granule neurons [10,28,29], neuroepithelial cells [4,27], dorsal root ganglia (DRG) neurons [11±13], and sympathetic neurons [17,18]. These PAs may facilitate Abbreviations: Bt2-cAMP, dibutyryl-cAMP; PA, plasminogen activator; uPA, urokinase; tPA, tissue plasminogen activator; DMEM, Dulbecco's modi®ed Eagle's medium; ITS+, Insulin, Transferrin, Selenium; BSA, linoleic acid media supplement. This article is dedicated to the memory of Antonia Vernadakis, PhD, whose numerous contributions to developmental neuroscience and her development of young neuroscientists had a major impact in expanding the ®eld of Developmental Neuroscience. * Corresponding author. Tel.: +1-303-315-5227; fax: +1-303-3153431. E-mail address: [email protected] (N.W. Seeds). p

granule cell migration [16,23], axonal growth [11,12,14,17], and synaptic plasticity [19]. Di€erentiating NG108-15 neuroblastoma cells synthesize elevated levels of tPA [9,12,26]. This secreted tPA is localized to the extending neurites, cell soma, and associated with the matrix underneath the soma and processes [12]. We show here that tPA mRNA expression can be induced in NG 108-15 cells by a number of di€erent growth conditions.

2. Experimental procedures Neuroblastoma NG 108-15 cells (3  105) were plated on 100 mm tissue culture dishes (Corning) in 10 ml of Dulbecco's modi®ed Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) (Hyclone; Logan, UT). After 24 h the plates were washed three times in Saline I and experimental media was added. The experimental media were either 10 ml of: DMEM+10% FBS; DMEM+10% FBS+1 mM dibutyryl-cAMP (Bt2cAMP); DMEM+1% FBS; DMEM+1% FBS+1 mM Bt2-cAMP; DMEM/F12+ITS+ (Collaborative Research Inc.); or DMEM/ F12+ITS++1 mM Bt2-cAMP. The cultures were

0736-5748/00/$20.00 7 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 5 7 4 8 ( 9 9 ) 0 0 0 8 2 - 9

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incubated at 378C in a 5% CO2 incubator for 15 or 24 h. NG 108-15 cells were also grown in suspension culture in 50 ml culture ¯asks at 378C in a shaking water bath at 125 rpm for 24 h. Total RNA was extracted as described by Chomczynski and Sacchi [2]. Northern blot analysis was performed with equal amounts of total RNA [22]. The RNA was transferred in 10  SSC from 1% agarose, 2.2 M formaldehyde gel to a ZetaProbe GT membrane (BioRad). Blots were hybridized in 15 ml of 0.25 M 32 PNa2POÿ 4 (pH 7.2) and 7% SDS, containing a 6 labelled mouse tPA cDNA probe (5  10 cpm/ml, 32  109 cpm/mg), at 658C for 24 h, and re-probed with a labeled probe to 18 S rRNA used to quantify the RNA loaded per lane. The blot was washed including

a ®nal wash of 0.02 M Na2POÿ 4 (pH 7.2) and 1% SDS at 658C. The blots were exposed and analyzed with a phosphoimager screen (Molecular Dynamics). Cells were photographed with Nikon 35 mm camera on an Olympus inverted microscope under a 10 phase-contrast objective using Kodak Plus-X pan ®lm. 3. Results When grown in DMEM and 10% FBS, NG 108-15 cells have a somewhat ¯attened morphology, and form small rapidly dividing colonies (Fig. 1A). The addition of 1 mM Bt2-cAMP causes the NG 108-15 cells to form larger colonies in which cells pile up on each

Fig. 1. NG108-15 Neuroblastoma di€erentiation. Cell cultures after 24 h in the indicated growth media: (A) DMEM+10% FBS; (B) DMEM+10% FBS+1 mM Bt2-cAMP; (C) DMEM+1% FBS; (D) DMEM+1% FBS+1 mM Bt2-cAMP; (E) DMEM/F12+ITS+; (F) DMEM/F12+ITS++Bt2-cAMP. Suspension cultures: (G) DMEM/F12+ITS+; (H) DMEM/F12+ITS++Bt2-cAMP.

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other (Fig. 1B). When grown in DMEM and 1% FBS, the NG 108-15 cells begin to morphologically di€erentiate (Fig. 1C) as indicated by the extension of short neuritic processes. The addition of 1 mM Bt2-cAMP enhances this di€erentiation, and the cells now extend long processes (Fig. 1D). When grown in DMEM/ F12+ITS+ the cells di€erentiate further (Fig. 1E). The addition of 1 mM Bt2-cAMP again enhances this di€erentiation and the NG 108-15 cells have extended elaborate neurites and appear more neuron-like than in any of the other growth conditions (Fig. 1F). When grown in suspension culture the NG 108-15 cells form large aggregates of cells (Fig. 1G and H). After 15 h of culture in the various growth media, Northern blot analysis reveals that tPA mRNA levels are increased under all conditions where 1 mM Bt2cAMP is added (Fig. 2). Since ribosomal RNA levels in these cells do not change with growth conditions over the short time course used in these studies, all blots were re-probed with an 18 S rRNA cDNA to quantify the RNA load per gel lane (only shown for Fig. 2). All the tPA mRNA values were normalized per 18 S rRNA per each lane and expressed relative to the level present in cells grown in DMEM+10% FBS (Table 1). NG 108-15 cells in 10% FBS with 1 mM Bt2-cAMP show a 4.1-fold increase in tPA mRNA

Fig. 2. Expression of tPA mRNA in di€erentiating NG108-15 cells after 15 h. Total RNA (30 mg) from indicated growth conditions applied to each lane and the blot hybridized with cDNA probe to tPA (upper panel), or 18 S rRNA (bottom panel). Lane (1) DMEM+10% FBS; Lane (2) DMEM+10% FBS+1 mM Bt2cAMP; Lane (3) DMEM+1% FBS; Lane (4) DMEM+1% FBS+1 mM Bt2-cAMP; Lane (5) DMEM/F12+ITS+; Lane (6) DMEM/F12+ITS++1 mM Bt2-cAMP.

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compared to cells grown in 10% FBS without Bt2cAMP. Cells in 1% FBS show 1.7-fold more tPA than those in 10% FBS and the addition of 1 mM Bt2cAMP gives a 3.5-fold increase. Similarly, cells grown in DMEM/F12 with the ITS+ supplement show a 1.3fold increase in tPA mRNA, while those grown in ITS+ with 1 mM Bt2-cAMP show a 3.6-fold increase in tPA mRNA. Extended time in culture shows that after 24 h tPA mRNA levels are further increased under all conditions as revealed by Northern blot analysis of the cells RNA (Fig. 3). When grown in 10% FBS the addition of Bt2-cAMP increased tPA mRNA 5.5-fold. In 1% FBS media tPA mRNA levels were 2.5-fold higher

Fig. 3. (a) Expression of tPA mRNA in di€erentiating NG108-15 cells after 24 h. Total RNA (30 mg) from indicated growth conditions applied to each lane and hybridized with a cDNA probe to tPA. Lane (1) DMEM+10% FBS; Lane (2) DMEM+10% FBS+1 mM Bt2-cAMP; Lane (3) DMEM+1% FBS; Lane (4) DMEM+1% FBS+1 mM Bt2-cAMP; Lane (5) DMEM/F12+ITS+; Lane (6) DMEM/F12+ITS++1 mM Bt2-cAMP. (b) Expression of tPA mRNA by NG108-15 neuroblastoma grown in suspension. Total RNA (10 mg) from the indicated growth conditions applied to each lane and the blot hybridized with a cDNA probe to tPA. Lane (1) DMEM+10% FBS; Lane (2) DMEM+10% FBS+1 mM Bt2cAMP; Lane (3) DMEM/F12+ITS+; Lane (4) DMEM/ F12+ITS++1 mM Bt2-cAMP.

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Table 1 Relative tPA mRNA levels in neuroblastoma NG108-15 cells grown under di€erent conditionsa Growth conditions

Time (h)

Attached

Suspension

DMEM+10% FBS DMEM+10% FBS+1 mM Bt2-cAMP DMEM+1% FBS DMEM+1% FBS+1 mM Bt2-cAMP DMEM/F12+ITS DMEM/F12+ITS+1 mM Bt2-cAMP DMEM+10% FBS DMEM+10% FBS+1 mM Bt2-cAMP DMEM+1% FBS DMEM+1% FBS+1 mM Bt2-cAMP DMEM/F12+ITS DMEM/F12+ITS+1 mM Bt2-cAMP

15 15 15 15 15 15 24 24 24 24 24 24

1.00 4.15 1.71 3.54 1.34 3.62 1.00 5.51 2.53 8.12 1.01 6.23

NA NA NA NA NA NA 5.10 9.03 NA NA 7.22 13.91

a

Northern Blots in Figs. 2 and 3 were exposed and analyzed with a phosphoimager screen (Molecular Dynamics). Density of the tPA band from cell RNA extracts grown under standard conditions (DMEM+10% FBS) and normalized for RNA loading was given a relative value of 1.00, to which all samples were compared. NA, not assessed under these conditions.

than the 10% FBS level. The addition of Bt2-cAMP increased tPA mRNA 3.2-fold over the 1% FBS level and 8-fold over the 10% FBS. In serum-free media the tPA mRNA level was about equal to the 10% FBS level at 24 h. However, cells cultured in ITS+ with Bt2-cAMP increased their expression 6.2-fold. Fig. 4 shows the tPA mRNA increases relative to cells cultured in DMEM with 10% FBS. These experiments show that conditions which induce tPA expression, i.e. low serum plus Bt2-cAMP, also induce extensive neurite outgrowth. This con®rms previous results on the induction of PA enzymatic activity [9,26] and shows that Bt2-cAMP induces transcription of tPA mRNA, probably through the CRE region of the tPA gene [20,21]. This elevation in tPA

mRNA levels translates into increased tPA activity secreted by these cells [12,25], that may facilitate the extension of neurites from di€erentiating NG108-15 cells. When grown in suspension NG108-15 cells form large aggregates under all growth conditions. Interestingly, suspension cultures in DMEM+10% FBS showed tPA mRNA levels 5-fold higher than those same cells grown on a tissue culture plastic surface (Fig. 3B). The addition of Bt2-cAMP increased the tPA mRNA level another 1.8-fold. In serum-free conditions NG108-15 cells in suspension showed tPA mRNA levels that were 7.2-fold higher than attached cells, and the level increased another 1.9-fold with the addition of Bt2-cAMP.

Fig. 4. Relative levels of tPA mRNA in NG108-15 neuroblastoma cells cultured under di€erent growth conditions.

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4. Discussion PA activity is associated with the extending axon of NG108-15 cells and regenerating peripheral neurons [7,8,12,18]; furthermore, some of this secreted PA activity is associated with the underlying matrix [12]. Mouse DRG sensory neurons in culture show an increase in PA mRNA levels coincident with axonal regeneration [7]. Furthermore, neurite outgrowth from peripheral neurons can be in¯uenced by the presence of PA inhibitors [5,6,18,24]. Protease nexin-1 at concentrations up to 30 ng/ml [24], and soybean trypsin inhibitor (STI) at concentrations up to 10 mg/ml [5,6] enhanced neurite outgrowth from DRG neurons. Higher concentrations of these inhibitors led to a decrease in neurite outgrowth. Furthermore, neurites grew preferentially towards a di€usable source of STI or grew onto an immobilized zone of STI (100 mg/ml), but away from zones with higher concentrations (>250 mg/ml) of STI. These results suggest that there is an optimum level of proteolytic activity necessary for neurite outgrowth [5,15], which is regulated by cells adjusting their levels of protease and protease inhibitors. Attachment to a solid matrix appears to regulate the level of tPA expression. uPA, the other PA produced by vertebrates, was induced in LLC-PK1 porcine kidney cells grown in suspension, or by disruption of the cytoskeleton with cytochalasin B, colchicine, or nocodazol [1]. When grown in suspension uPA levels were further induced by the addition of 8-bromo-cAMP, similar to the ®ndings we observed for tPA in NG10815 cells. These results suggest that PA production is regulated through the cytoskeleton, and may occur by the interaction of integrin receptor molecules with their extracellular matrix ligand. Evidence for an integrin response is seen in the production of uPA by endocardial cells. When grown on the 45-kDa gelatin binding fragment of ®bronectin, uPA production by endocardial cells increased 3-fold as compared to growth on native ®bronectin [13]. The morphology of these cells growing on the 45-kDa ®bronectin fragment was more fusiform compared to the ¯attened morphology when grown on other matrices. Currently, we are investigating the e€ects of various extracellular matrix molecules on the di€erentiation and tPA production by NG108-15 cells. Furthermore, we are also exploring the e€ect of cytoskeleton perturbation on tPA production by these cells.

Acknowledgements We thank Ms Darcy Williams for her technical support. These studies were supported by grants from the

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