Ž .Developmental Brain Research 99 1997 53–60

Research report

Effects of 1,25-dihydroxyvitamin D on growth of mouse neuroblastoma3

cells

Timothy D. Veenstra a, James M. Londowski a, Anthony J. Windebank c, Stephen Brimijoin d,Rajiv Kumar a,b,)

a Nephrology Research Unit, Mayo Clinic Foundation, 200 First St., SW, 911A Guggenheim Bldg., Rochester, MN 55905, USAb Departments of Medicine, Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, MN 55905, USA

c Department of Neurology, Mayo Clinic Foundation, Rochester, MN 55905, USAd Department of Pharmacology, Mayo Clinic Foundation, Rochester, MN 55905, USA

Accepted 29 October 1996

Abstract

Ž Ž . .Epitopes of the 1,25-dihydroxyvitamin D 1,25 OH D receptor have been shown in developing dorsal root ganglia in fetal mice, as2 3wwell as in cells maintained in culture Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-Dihydroxyvitamin D receptors3

Ž . x Ž .in developing dorsal root ganglia of fetal rats, DeÕ. Brain Res., 92 1996 120–124 . To investigate a possible role for 1,25 OH D in2 3Ž .neural cell growth and development, a murine neuroblastoma cell line that expresses 1,25 OH D receptors, was treated with2 3

Ž . Ž .1,25 OH D . Treatment with 1,25 OH D resulted in a decrease in cell proliferation, a change in cell morphology, and the expression2 3 2 3

of protein markers of mature neuronal cells. The decrease in cell proliferation was accompanied by an increase in the expression of nerveŽ .growth factor NGF . Anti-NGF monoclonal antibody added to the growth medium blocked the decrease in cell proliferation caused by

Ž . Ž .1,25 OH D treatment. Our results show that the sterol hormone 1,25 OH D , causes a decrease in the proliferation of mouse2 3 2 3

neuroblastoma cells through alterations in the expression of NGF.

Keywords: 1,25-Dihydroxyvitamin D ; Neuroblastoma; Decrease in proliferation; NGF3

1. Introduction

Ž Ž . .1,25-Dihydroxyvitamin D 1,25 OH D , the active3 2 3

metabolite of vitamin D, is known to regulate epithelialcalcium transport, cellular growth, and the transcription of

w xseveral genes 4,8,20,23,24,31,44,45 . 1,25-Dihydroxy-vitamin D exerts it biological effects primarily through3

Ž . Ž . w xintracellular 1,25 OH D receptors VDR 13,30 . Im-2 3

munohistochemical studies have located epitopes for theVDR in several tissues of the developing fetus. The VDRhas been detected in developing dorsal root ganglia in vivo

w xand in dorsal root ganglion cells maintained in culture 18 ,Ž .suggesting 1,25 OH D plays an as yet undetermined role2 3

in cellular growth, development, or function in the nervoussystem. In addition, the VDR has been detected in devel-

w xoping fetal bone and kidney 17 , once again raising the

) Ž .Corresponding author. Fax: q1 507 266-4710. E-mail:rkumar@mayo.edu

Ž .possibility that 1,25 OH D plays a role in fetal develop-2 3Ž .ment. It is of interest that plasma 1,25 OH D concentra-2 3

tions are increased very early in pregnancy, well beforeincreases in intestinal calcium transport occur, suggesting a

w xrole for the sterol in normal fetal development 21,22 .Ž .1,25 OH D has been shown to regulate the expres-2 3

w x w xsion of NGF in osteoblasts 14 , fibroblasts 43 and glialw xcells 27 maintained in culture; however, the role of

Ž .1,25 OH D in altering growth and development via2 3

NGF-mediated mechanisms has not been investigated. NGFis a member of the neurotrophin family which includes

Ž .brain-derived neurotrophic factor BDNF , and neu-Ž .rotrophins 3, 4, and 5 NT-3, NT-4, and NT-5 and has

been shown to be required for the development and main-w xtenance of sympathetic and sensory neurons 38 .

Widespread distribution of NGF or NGF receptor mRNAsŽ .throughout the brain and central nervous system CNS

suggest that NGF also plays a physiological role withinw xthese systems 17,35 . It is well documented that the

treatment of the clonal rat pheochromocytoma cell line

0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 96 00196-4

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–6054

PC12 with NGF induces these cells to differentiate asshown by a decrease in cell proliferation accompanied by

w xthe growth of neurite extensions 12 , an increase in elec-w xtrical excitability 10 , and a change in neurotransmitter

w xsynthesis 35 .Ž .We show that 1,25 OH D causes a dose-dependent2 3

decrease in cell proliferation in mouse N1E-115 neuroblas-Ž .toma cells. Treatment of N1E-115 cells with 1,25 OH D2 3

also results in an increase in the expression of NGF, andŽ .the effects of 1,25 OH D on the proliferation of the cells2 3

are reversed when mouse anti-NGF monoclonal antibodyis added to the cell media. These results suggest the effects

Ž .of 1,25 OH D on mouse neuroblastoma cells are medi-2 3

ated through a NGF-directed pathway.

2. Materials and methods

2.1. Materials

N1E-115 clones derived from mouse neuroblastomaw xC1300 cell line 2 were the generous gift of Dr. E.

Ž .Richelson Mayo Clinic, Scottsdale, AZ . Mouse mono-clonal anti-tau-1 antibody, mouse monoclonal anti-nerve

Ž .growth factor NGF antibody, monoclonal anti-NGF con-jugated with b-galactosidase antibody, mouse NGF, and

Ž .chlorphenolred-b-D-galactopyranoside CPRG were ob-Ž .tained from Boehringer Mannheim Indianapolis, IN .

Mouse monoclonal anti-GAP-43 antibody was obtainedŽ .from Sigma St. Louis, MO .

2.2. Culture and growth of N1 E-115 cells

Ž .Murine neuroblastoma cells N1E-115, C1300 weregrown in Dulbecco’s modified Eagle’s medium supple-

Ž .mented with 10% fetal bovine serum FBS , 7.3 mgrlbiotin, 0.002 grl thioctic acid, and 0.00136 grl vitaminB . For differentiation by serum withdrawal, cells were12

cultured in the above medium without FBS. The N1E-115cells were seeded at 1500 cells per well in 24-well plates,24 h before an experiment. Four wells of each plate weretreated on a daily basis with 10y12 M, 10y10 M, or 10y8

Ž .M 1,25 OH D . Four wells of cells were serum-deprived.2 3

Eight wells of each plate acted as controls and were treatedŽ .with vehicle ethanol . The growth medium was removed

daily and replaced with fresh medium containingŽ .1,25 OH D or vehicle. Cells were lifted from each well2 3

Žby removing the medium and adding 1 ml of PUCKS 1.0g glucose, 20 g sucrose, 8 g NaCl, 0.4 g KCl, 45 mg

.Na HPO , 30 mg KH PO per 100 ml H 0 to each well2 4 2 4 2

and incubating at 378C for 15 min. The plates werecarefully examined under a microscope to ensure that thecells had been removed from the wells. The cell suspen-sion was added to 19 ml of Isoton II and the numbers ofcells per well were determined with a Coulter counterŽ .Coulter Electronics Inc., Hialeah, FL . In experiments

where N1E-115 cell proliferation was measured in theŽpresence of murine monoclonal anti-NGF antibody Boeh-

.ringer Mannheim , the conditions were identical to thosedescribed above except that 40 ngrml of murine mono-clonal anti-NGF antibody was added to each treatment.

To determine the percentage of cells that had undergoneŽ .a morphological change in response to 1,25 OH D , cells2 3

from 9–10 random areas within the plate wells wereclassified as round, flattened out, or showed some evi-dence of neurite extension. Cells were photographed andgraded by an independent member of the laboratory whowas blinded to the treatment of the cells. Only cells thathad flattened out or had extended neurites were consideredto be morphologically altered.

2.3. Enzyme-linked immunosorbent assay to measure theexpression of tau-1 protein

N1E-115 cells were plated out into seven T-175 cellculture flasks at a density of 5=105 cells per flask andallowed to grow to confluency. Three flasks were treated

y8 Ž .on a daily basis with 10 M 1,25 OH D , and three2 3

were treated with ethanol. As before, the cells receivedfresh medium with the appropriate treatment on a dailybasis. Total cellular protein was isolated from one flask ofeach treatment on a daily basis by lifting the cells, andcentrifuging them at 1000=g for 3 min. The cell pelletwas washed three times with PBS, pH 7.4. The cell pellet

Žwas then resuspended in 1.0 ml of cell lysis buffer 1%Triton X-100, 20 mM HEPES, 150 mM NaCl, 2 mM

.CaCl supplemented with 10 mgrml PMSF, 1 mgrml2

aprotinin, and 1 mgrml leupeptin. The cell suspension waskept on ice for 2 h and periodically vortexed during thisperiod. The suspension was then centrifuged at 14,000 rpmat 48C for 15 min to pellet cell debris. The supernatant wastransferred to a fresh microcentrifuge tube and freeze driedovernight. The following day the lyophilized protein was

Žresuspended in carbonate coating buffer 1.6 mg Na CO ,2 3.3.0 mg NaHCO , 0.2 mg NaN , per ml of H 0 and the3 3 2

protein concentration of each sample was measured usingw xthe Bio-Rad protein assay 3 . 200 ml of each of the above

protein aliquots were placed into the wells of an ELISAmicrotitre plate. The plate was left overnight at roomtemperature. The following day each well was washed 5times with PBS, 0.05% Tween-20, pH 7.4 followed by theaddition of 200 ml of a 1:500 dilution of anti-tau 1 mousemonoclonal antibody in PBS, pH 7.4. The antibody wasallowed to bind to the antigen at room temperature for 2 h.Excess antibody was removed by 5 washings with PBS,0.05% Tween-20, pH 7.4 after which 100 ml of anti-mousesecondary antibody diluted 1:1000 in PBS, pH 7.4 wasadded to each well for 1 h at room temperature. 100 ml of

Ž Ždeveloping solution 5 mg of p-nitrophenyl phosphate in.5 ml of substrate buffer was added to each well and left to

incubate at room temperature for 30 min. Color develop-ment reaction was stopped by adding 100 ml of 3 M

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–60 55

NaOH to each well. The absorbance of each well at 405nm was recorded using a microtitre plate reader. Ab-sorbance was converted to absorbancermg of protein basedon the amount of protein.

2.4. Immunostaining for tau-1 and GAP-43 protein

N1E-115 cells were plated out onto cell culture slidesand allowed to attach overnight. Subsequently the cellswere treated daily with vehicle, or 10y12 M, 10y10 M, or

y8 Ž .10 M 1,25 OH D or were serum-deprived. A slide of2 3

each treatment was fixed daily by immersing the slide for5 min in ice-cold acetone followed by 10% formalin in 1%zinc sulfate for 30 min just before immunostaining. Sec-tions were then treated with 1% normal goat serum in PBSand 0.3% Triton X-100 for 10 min, followed by 3 washeswith PBS. Cell cultures were incubated with the anti-tau-1

Žprimary antibody for 60 min mouse-anti-tau-1, 5 mgrml.in PBS containing 1% normal goat serum at room temper-

ature. After thorough rinsing, all sections were treated withŽbiotinylated goat anti-mouse immunoglobulin G IgG,

.1:400; Dako, Carpinteria, CA , followed by peroxidase-Ž .labeled streptavidin Dako, 1:500 for 30 min at room

temperature. Sections were developed for 15 min by adding0.1 M sodium acetate, pH 5.2 containing aminoethylcar-bazole and H O . Coverslips were attached using aqueous2 2

mounting media.For staining with the anti-GAP-43 antibody, N1E-115

cells were plated on cell culture slides in normal growthmedium and allowed to attach overnight. Cells were treated

y8 Ž .daily with ethanol, or 10 M 1,25 OH D , or were2 3

serum-deprived. A slide of each treatment was fixed dailyusing the procedure above. Cell cultures were stainedusing the same procedure given for the anti-tau-1 stainingabove with a dilution of anti-GAP-43 protein primaryantibody of 1:400.

2.5. Enzyme-linked immunosorbent assay to measure ex-pression of nerÕe growth factor

N1E-115 cells were plated out into T-75 cell cultureflasks at a density of 150,000 cells per flask, and allowedto proliferate in growth medium with 10% FBS for 4 days,with the medium changed daily. After this time the mediumwas removed from one of the flasks and frozen at y208C.Of the remaining flasks, 6 were treated with 10y8 M

Ž .1,25 OH D and 6 were treated with ethanol. At time2 3

intervals of 3, 6, 12, 24, 48, and 72 h, media was removedfrom each of the trials and frozen at y208C. The mediumwas replaced after 24 and 48 h. The total number of cellsin each flask was also recorded at each time interval. NGFexpression was measured by coating a 96-well microtitreplate with 80 ngrml murine monoclonal anti-NGF dis-

Žsolved in coating buffer 50 mM Na CO rNaHCO , 0.1%2 3 3Ž . .wrv NaN , pH 9.6 , and incubating the plate at 378C for3

2 h. After this time the coating buffer was removed and

Ž Ž .200 ml of blocking solution 0.5% wrv BSA, in coating.buffer was added to each well and the plate incubated at

378C for 30 min. The blocking solution was removed andŽthe wells were washed three times with wash buffer 50

Ž .mM Tris-HCl, 200 mM NaCl, 10 mM CaCl , 0.1% wrv2Ž . .Triton X-100, 0.05% wrv NaN , pH 7.0 . After wash-2

ing, 100 ml of each sample was applied to the appropriatewells and the plate incubated overnight at 48C. The follow-ing day the wells were washed three times with washbuffer and 100 ml of murine monoclonal anti-NGF b-

Ž .galactosidase conjugated antibody 0.4 Urml was addedto each well. The plate was incubated at 378C for 4 h.After the plate was washed three times, 200 ml of sub-

Žstrate solution 40 mg of chlorophenolred-b-D-galacto-pyranoside dissolved in 20 ml of 100 mM Hepes, 150 mM

Ž . Ž .NaCl, 2 mM MgCl , 1% wrv BSA, 0.1% wrv NaN ,2 3.pH 7.0 was added to each well. The plate was placed at

378C for 30 min to develop. The absorbance of each well

Ž .Fig. 1. A: effect of 1,25 OH D on the proliferation of N1E-115 cells.2 3

N1E-115 cells were grown in growth medium with serum treated withŽ . Ž . y1 2 Ž . y1 0 Ž . y8 Ž .ethanol control I , 10 M B , 10 M ` , 10 M v

Ž . Ž .1,25 OH D , or serum-deprived ' . B: effect of anti-NGF antibody on2 3Ž . Ž .the proliferation of N1E-115 cells treated with ethanol control I ,

y1 2 Ž . y1 0 Ž . y8 Ž . Ž .10 M B , 10 M ` , 10 M v 1,25 OH D , or serum-de-2 3Ž .prived ' . Data are the mean"S.E.M.

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–6056

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–60 57

was measured at 570 nm. The final values were calculatedas pg of NGF per ml per 106 cells.

3. Results

Using immunohistochemistry, epitopes for the VDR areobserved within the neuroblastoma cell line, N1E-115Ž .data not shown , as they are in the dorsal root ganglia

w xcells maintained in culture 18 . In order to elucidate theŽ .role of 1,25 OH D in neural cells, this cell line, was2 3

Ž .treated with 1,25 OH D on a daily basis and the effects2 3

of the sterol on cell growth were measured. Cells treatedŽ .with 1,25 OH D showed a dose-dependent decrease in2 3

Ž .cell proliferation Fig. 1A . The decrease in cell prolifera-tion was evident within 24 h of treatment. Neuroblastoma

Ž .cells treated with 1,25 OH D showed morphological2 3

changes which included flattening out of the cells and theŽappearance of short protrusions from the cell body Fig.

.2A . Vehicle treated cells did not show correspondingŽ .changes Fig. 2B . In general, a 15-fold increase in the

number of cells exhibiting a morphological change wasŽ .observed in 1,25 OH D treated cells compared to control2 3

cells. Cells that were serum deprived, a maneuver knownto cause growth arrest and differentiation in this cell line,did not proliferate and differentiated as determined by theextension of long neurites protruding from the cell.

To determine if the decrease in proliferation observed inŽ .the N1E-115 cells treated with 1,25 OH D is associated2 3

with cell differentiation, cells were examined for an in-crease in the expression of protein markers indicative ofmature, differentiated cells. In neuroblastoma N1E-115cells, these protein markers include tau-1, growth associ-

Ž .ated protein 43 GAP-43 , and neurofilament proteinsw x11,29,42 . An increase in the expression of tau-1 proteinwas observed using an enzyme-linked immunosorbent as-

Ž . Ž .say ELISA Fig. 3 . The increase in the expression oftau-1 was observed within 24 h of treating the cells with

y8 Ž .10 M 1,25 OH D and was consistently greater than2 3

the tau-1 expression in the control cells. In order toconfirm the increase in the expression of tau-1 in

Ž .1,25 OH D treated cells and also examine for a change2 3

in the expression of GAP-43, N1E-115 cells were platedonto cell culture slides, fixed and immunostained usingmonoclonal antibodies to tau-1 and GAP-43. An increasein the expression of both tau-1 and GAP-43 was observedin cells treated with 10y8 , 10y10, and 10y12 M

Ž .1,25 OH D using immunohistochemical techniques when2 3Ž .compared to cells treated with vehicle data not shown .

Ž .To elucidate a mechanism by which 1,25 OH D2 3

causes a decrease in the proliferation of N1E-115 cells, the

Fig. 3. ELISA measuring the expression of tau-1 protein in N1E-115Ž . y8 Ž . Ž .control cells I and cells treated with 10 M 1,25 OH D B . Data2 3

are the mean"S.E.M. values of three separate experiments.

expression of NGF was measured in cells treated withy8 Ž .10 M 1,25 OH D using a sensitive sandwich ELISA.2 3

NGF has been shown to be required for the developmentw xand maintenance of sympathetic and sensory neurons 41

and NGF and NGF receptor mRNAs have been shown tow xbe widely distributed throughout the brain and CNS 19,38 .

y8 Ž .In cells treated with 10 M 1,25 OH D a 10-fold2 3

increase in NGF expression was observed within 3 h ofŽ .treatment Fig. 4 . The levels of NGF expression decreased

over the course of the experiment, but were still greatlyenhanced when compared to the control cells. A decreasewas observed in the concentration of NGF in the untreatedcells suggesting the endogenous NGF in the medium wasbeing degraded as the cell numbers increased over time.To determine the correlation between the increased NGFexpression in the differentiation of the cells by

Ž .1,25 OH D , the cell proliferation experiments were re-2 3

peated, in the presence of 40 ngrml of murine monoclonalŽ .anti-NGF antibody Fig. 1B . In the presence of anti-NGF

antibody, cells treated with 10y12 M and 10y10 MŽ .1,25 OH D proliferated at the same rate as the control2 3

y8 Ž .cells. Cells treated with 10 M 1,25 OH D grew2 3

slightly slower than the control cells during days 4 and 5,but were obviously proliferating at a much greater rate

Žthan in the absence of anti-NGF antibody compare with. Ž .Fig. 1A . The numbers of cells in the 1,25 OH D -treated2 3

wells were very similar to the number of cells in thecontrol wells at the completion of the experiment. In thepresence of anti-NGF antibody, there was no change in thedifferentiation of serum deprived cells. No obvious changes

y8 Ž . Ž .Fig. 2. A: morphology changes observed in N1E-115 cells treated with 10 M 1,25 OH D for 5 days compared to B control cells treated with ethanol2 3y8 Ž .for 5 days. C: morphology changes in N1E-115 cells grown in the presence of 40 ngrnl anti-NGF polyclonal antibody treated with 10 M 1,25 OH D2 3

Ž .for 5 days compared to D control cells treated with ethanol for 5 days.

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–6058

Ž .Fig. 4. Sandwich ELISA for expression of NGF in 1,25 OH D treated2 3

N1E-115 neuroblastoma cells. N1E-115 cells were maintained for 72 h ingrowth medium with serum and subsequently treated with 10y8 M

Ž . Ž . Ž .1,25 OH D B or ethanol I . The medium was removed and tested2 3

for the presence of NGF using a sandwich ELISA at the indicated timepoints.

in the morphology of the cells were observed in theŽ .1,25 OH D -treated cells even after 5 days of treatment2 3

Ž .Fig. 2C,D . The lack of a significant decrease in cellproliferation and a lack of change in morphology whenanti-NGF antibody is added to the growth medium, sug-

Ž .gests that 1,25 OH D causes a decrease in the prolifera-2 3

tion of neuroblastoma cells via a NGF-mediated pathway.Cultures of N1E-115 cells immunostained for the presence

Ž .of the high and low affinity NGF receptors TrkA and p75showed that both types of NGF receptors were present in

Ž .this cell line data not shown .

4. Discussion

Ž .Our results show that 1,25 OH D induces a decrease2 3

in the proliferation of mouse neuroblastoma N1E-115 cells.Not only was a significant decrease in cell proliferationalong with a change in cell morphology seen in cells

Ž .treated with 1,25 OH D , but an increase in the levels of2 3

tau-1 and GAP-43 protein, known markers of differenti-w xated neuronal cells 11,42 , was also observed. The results

Ž .also suggest that 1,25 OH D alters the growth rate of2 3

these cells by increasing the expression of NGF, as theŽ .effects of 1,25 OH D on these cells could be blocked by2 3

the addition of murine monoclonal anti-NGF antibody tothe growth media.

Ž .1,25 OH D has been shown to induce NGF synthesis2 3w x w xin primary cultures of glial cells 29 , fibroblasts 46 , and

w xosteoblasts 16 . In all three cell lines, an increase wasobserved in NGF mRNA levels, showing the effects of

Ž .1,25 OH D on NGF synthesis are pre-translational. In2 3

Ž .the case of osteoblastic cells, 1,25 OH D was also shown2 3w xto increase the expression of BDNF 16 . Not only does

Ž .our study show that 1,25 OH D increases NGF expres-2 3

sion, but it also shows that neutralizing the NGF activityusing a monoclonal anti-NGF antibody blocks the anti-pro-

Ž .liferative effects of 1,25 OH D . This suggests that en-2 3

hanced NGF expression plays a direct role in theŽ .1,25 OH D mediated growth arrest of neuroblastoma2 3

cells. Although our results do not provide a direct mecha-Ž .nism by which 1,25 OH D induces NGF expression, it is2 3

Ž .possible that 1,25 OH D , acting through the VDR, may2 3

directly promote the expression of the NGF gene. Theproto-oncogenes c-fos and c-jun have been shown to beinvolved in NGF-gene regulation via a phorbol ester-re-

w xsponsive element 1,32,34 ‘TGAGTCA’ present in thew xfirst intron of the NGF gene 14,27,47 . A very similar

phorbol ester-responsive element ‘TGACTCA’ is recog-Ž .nized by retinoic acid and 1,25 OH D receptors and the2 3

w xFos-Jun complex in the osteocalcin gene 35 . Studies areongoing in an attempt to establish the mechanism by

Ž .which 1,25 OH D enhances NGF expression.2 3Ž .The precise manner in which 1,25 OH D alters growth2 3

Ž .in other non-neuronal cells not known. 1,25 OH D is2 3

known to alter the expression of insulin-like growth factorsŽ . w xIGFs 5,6,28,34 , insulin-like growth factor binding pro-

Ž . w x w xteins IGF-BPs 6,26 , nerve growth factor 16,47 , andw xthe expression of growth factor receptors 9,25 in variousŽ .non-neuronal types of cells. Thus, 1,25 OH D could2 3

influence growth factor synthesis and alter cell growth inan autocrine or paracrine manner. Administration of

Ž .1,25 OH D has also been shown to increase Raf kinase2 3

activity and to shift Raf from a cytoplasmic to a perinu-w xclear region 26 . Antiproliferative effects on human kera-

tinocytes, accompanied by an inhibition of c-myc, havew xbeen reported in human keratinocytes 36,37 , MCF-7 and

w xT47D human breast cancer cells 33 , and myelo-mono-w x Ž .cytic leukemia cells 7 treated with 1,25 OH D . c-Ki-ras2 3

w xmRNA is increased in chondrocytes 15 , and proteinkinase C activity is increased in diverse cell typesw x Ž .39,40,43 . Thus it is also possible that 1,25 OH D could2 3

influence cell growth by influencing the expression andactivity of intracellular growth regulatory proteins. We are

Ž .presently investigating the effect of 1,25 OH D on the2 3

expression of various cell cycle regulatory molecules in anattempt to better understand the possible role of

Ž .1,25 OH D in the growth and differentiation of neurob-2 3

lastoma cells.Ž .In conclusion, we have shown that 1,25 OH D causes2 3

a decrease in the proliferation of murine N1E-115 neurob-lastoma cells. The decrease in proliferation of the cells

Ž .caused by 1,25 OH D is accompanied by a change in2 3

cell morphology, an increased expression of protein mark-ers of mature neuronal cell, and an increase in the expres-sion of NGF. Furthermore, we have shown that the anti-

Ž .proliferative morphological effects of 1,25 OH D di-2 3

rectly involves the enhanced expression of NGF.

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–60 59

Acknowledgements

We would like to thank Steven C. Zeismer for expertassistance in performing the immunohistochemical tech-

Ž .niques. Supported by NIH Grants: DK25409 R.K. ,Ž .DK42971 R.K.

References

w x1 Angel, P., Imagawa, M., Chice, R., Stein, B., Imbra, R.J., Rahms-dorf, H.J., Jonat, C., Herrlich, P. and Karin, M., Phorbol ester-in-ducible genes contain a common cis element recognized by a

Ž .TPA-modulated trans -acting factor, Cell, 49 1987 729–739.w x2 Augusti-Tocco, G. and Sato, G., Establishment of functional clonal

lines of neurons from mouse neuroblastoma, Proc. Natl. Acad. Sci.Ž .USA, 69 1969 258–263.

w x3 Bradford, M., A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye

Ž .binding, Anal. Biochem., 72 1976 248–254.w x4 Cai, Q., Chandler, J.S., Wasserman, R.H., Kumar, R. and Penniston,

J.T., Vitamin D and adaptation to calcium and phosphate deficien-cies increase intestinal plasma membrane calcium pump gene ex-

Ž .pression, Proc. Natl. Acad. Sci. USA, 90 1993 1345–1349.w x5 Chen, T.L., Mallory, J.B. and Hintz, R.L., Dexamethasone and

1,25-dihydroxyvitamin D modulate the synthesis of insulin-like3Ž .growth factor-I in osteoblast-like cells, Calcif. Tiss. Int., 48 1991

278–282.w x6 Chen, T.L., Chang, L.Y., Bates, R.L. and Perlman, A.J., Dexametha-

sone and 1,25-dihydroxyvitamin modulation of insulin-like growthfactor-binding proteins in rat osteoblast-like cell cultures, En-

Ž .docrinology, 128 1991 73–80.w x7 DeLuca, H.F., The relationship between the vitamin D system and

Ž .cancer, AdÕ. Exp. Med. Biol., 206 1986 413–429.w x8 DeLuca, H.F., The vitamin D story: a collaborative effort of basic

Ž .science, and clinical medicine, FASEB J., 2 1988 3043–3053.w x9 Desprez, P.Y., Poujol, D., Falette, N., Lefebvre, M.F. and Saez, S.,

Ž .1,25 OH D increases epidermal growth factor receptor gene ex-2 3

pression in BT-20 breast carcinoma cells, Biochem. Biophys. Res.Ž .Commun., 176 1991 1–6.

w x10 Dichter, M.A., Tischler, A.S. and Green, L.A., Nerve growthfactor-induced increase in electrical excitability and acetylcholine

( )senstivity of a rat pheochromocytoma cell line, Nature London ,Ž .268 1977 501–504.

w x11 Goslin, K., Schreyer, D.J., Skene, J.H.P. and Banker, G., Changes inthe distribution of GAP-43 during the development of neuronal

Ž .polarity, J. Neurosci., 10 199 588–602.w x12 Green, L.A. and Tischler, A., Establishment of a nonadrenergic

clonal line of rat adrenal pheochromocytoma cells which respond toŽ .nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 73 1976

2424–2428.w x13 Haussler, M.R., Mangelsdorf, D.J., Komm, B.S., Terpening, C.M.,

Yamaoka, K., Allegretto, E.A., Baker, A.R., Shine, J., McDonnell,D.P. and Hughes, M., Molecular biology of the vitamin D hormone,

Ž .Rec. Prog. Horm. Res., 44 1988 263–305.w x14 Hengerer, B., Lindholm, D., Heumann, R., Ruther, U. and Wagner,¨

E.F., Lesion-induced increase in nerve growth factor is mediated byŽ .c-fos, Proc. Natl. Acad. Sci. USA, 87 1990 3899–3903.

w x15 Huh, N., Satoh, M., Nose, K., Abe, E., Suda, T., Rajewsky, M.F.Ž .and Kuroki, T., 1a ,25 OH D induces anchorage-independent2 3

growth and c-Ki-ras expression of BALBr3T3 and NIHr3T3 cells,Ž .Jpn. J. Cancer Res., 78 1987 99–102.

w x16 Jehan, F., Naveilhan, P., Neveu, I., Harvie, D., Dicou, E., Brachet,P. and Wion, D., Regulation of NGF, BDNF and LNGFR gene

Ž .expression in ROS 17r2.8 cells, Mol. Cell. Endocrin., 116 1996149–156.

w x17 Johnson, J.A., Grande, J.P., Roche, P.C. and Kumar, R., Ontogenyof the 1,25-dihydroxyvitamin D receptor in fetal rat bone. J. Bone.3

Ž .Miner. Res., 11 1996 56–61.w x18 Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-

Dihydroxyvitamin D receptors in developing dorsal root ganglia of3Ž .fetal rats, DeÕ. Brain Res., 92 1996 120–124.

w x19 Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen,H., Levels of nerve growth factor and its mRNA in the centralnervous system of the rat correlate with cholinergic innervation,

Ž .EMBO J., 4 1985 1389–1393.w x20 Kumar, R., Schnoes, H.K. and DeLuca, H.F., Rat intestinal 25-hy-

droxyvitamin D and 1a ,25-dihydroxyvitamin D -24-hydroxylase,3 3Ž .J. Biol. Chem., 253 1978 3804–3809.

w x21 Kumar, R., Cohen, W.R., Silva, P. and Epstein, F.H., Elevated1,25-dihydroxyvitamin D plasma levels in normal human pregnancy

Ž .and lactation, J. Clin. InÕest., 63 1979 342–344.w x22 Kumar, R., Cohen, W.R. and Epstein, F.H., Vitamin D and calcium

Ž .hormones in pregnancy, N. Engl. J. Med., 302 1980 1143–1144.w x23 Kumar, R., Wieben, E. and Beecher, S.J., The molecular cloning of

the cDNA for bovine vitamin D-dependent calcium-binding protein:structure of the full-length protein and evidence for homologies withother calcium-binding proteins of the troponin-C superfamily of

Ž .proteins. Mol. Endocrinol., 3 1989 427–432.w x Ž .24 Kumar, R., Vitamin D and calcium transport, Kidney Int., 40 1991

1177–1189.w x25 Kurose, H., Yamaoka, K., Okada, S., Nakajima, S. and Seino, Y.,

Ž .1,25 OH D increases IGF-I receptors in clonal osteoblastic cells.2 3Ž .Study on interaction of IGF-I and 1,25 OH D , Endocrinology,2 3

Ž .126 1990 2088–2094.w x26 Lissoos, T.W., Beno, D.W. and Davis, B.H., 1,25-Dihydroxyvitamin

D activates Raf kinase and Raf perinuclear translocation via a3Ž .protein kinase C-dependent pathway, J. Biol. Chem., 268 1993

25132–25138.w x27 Mocchetti, I., De Bernardi, M.A., Szekely, A.M., Alho, H., Brooker,

G. and Costa, E., Regulation of nerve growth factor biosynthesis byb-adrenergic receptor activation in astrocytoma cells: a potential role

Ž .of c-Fos protein, Proc. Natl. Acad. Sci. USA, 86 1989 3891–3895.w x28 Moriwake, T., Tanaka, H., Kanzaki, S., Higuchi, J. and Seino, Y.,

1,25-Dihydroxyvitamin D stimulates the secretion of insulin-like3

growth factor binding protein 3 by cultured human osteosarcomaŽ .cells, Endocrinology, 130 1992 1071–1073.

w x29 Neveu, I., Naveilhan, P., Jehan, F., Baudet, C., Wion, D., DeLuca,H.F. and Brachet, P., 1,25-Dihydroxyvitamin D regulates the syn-3

thesis of nerve growth factor in primary cultures of glial cells, Mol.Ž .Brain Res., 24 1994 70–76.

w x30 Norman, A.W., Nemere, I., Zhou, L.X., Bishop, J.E., Lowe, K.E.,Ž .Maiyar, A.C., Collins, E.D., Taoka, T. and Sergeev, I., 1,25 OH -3

vitamin D , a steroid hormone that produces biological effects via3

both genomic and nongenomic pathways, J. Steriod Biochem. Mol.Ž .Biol., 41 1992 231–240.

w x31 Pike, J.W., Vitamin D3 receptors: structure and function in transcrip-Ž .tion, Annu. ReÕ. Nutr., 11 1991 189–216.

w x32 Sassone-Corsi, P., Lamph, W.W., Kamps, M. and Verma, I.M.,fos-Associated cellular p39 is related to nuclear transcription factor

Ž .AP1, Cell, 54 1988 553–560.w x33 Saunders, D.E., Christensen, C. and Wappler, N.L., Inhibition of

c-myc in breast and ovarian carcinoma cells by 1,25-dihydroxy-vitamin D , retinoic acid and dexamethasone, Anti-Cancer Drugs, 43Ž .1993 201–208.

w x34 Scharla, S.H., Strong, D.D., Mohan, S., Baylink, D.J. and Linkhart,T.A., 1,25-Dihydroxyvitamin D differentially regulates the produc-3

tion of insulin-like growth factor-I and IGF-4 in mouse osteoblasts,Ž .Endocrinology, 129 1991 3139–3146.

w x35 Schubert, D., Heinemann, S. and Kidokoro, Y., Cholinergicmetabolism and synapse formation by a rat nerve cell line, Proc.

Ž .Natl. Acad. Sci. U.S.A., 74 1977 2579–2582.w x36 Schule, R., Umesono, K., Mangelsdorf, D.J., Bolado, J., Pike, J.W.¨

( )T.D. Veenstra et al.rDeÕelopmental Brain Research 99 1997 53–6060

and Evans, R.M., Jun-Fos and receptors for vitamin A and Drecognize a common response element in the human osteocalcin

Ž .gene, Cell, 61 1990 497–504.w x37 Sebag, M., Henderson, J., Rhim, J. and Kremer, R., Relative resisi-

tance to 1,25-dihydroxyvitamin D in a keratinocyte model of tumor3Ž .progression, J. Biol. Chem., 267 1992 12162–12167.

w x38 Senut, M.C., Lamour, Y., Lee, J., Brachet, P. and Dicou, E.,Neuronal localization of the nerve growth factor precursor-like

Ž .immunoreactivity in the rat brain, Int. J. DeÕ. Neurosci., 8 199065–80.

w x39 Simboli-Campbell, M., Gagnon, A., Franks, D.J. and Welsch, J.,Ž .1,25 OH D translocates protein kinase Cb to nucleus and en-2 3

hances plasma membrane association of PKC in renal epithelialŽ .cells, J. Biol. Chem., 269 1994 3257–3264.

w x40 Sylvia, V.L., Schwartz, Z., Schuman, L., Morgan, R.T., Mackey, S.,Gomez, R. and Boyan, B.D., Maturation-dependent regulation ofprotein kinase C activity by vitamin D in chondrocyte cultures, J.3

Ž .Cell Physiol., 157 1993 271–278.w x41 Thoenen, H. and Barde, Y.A., Physiology of nerve growth factor,

Ž .Physiol. ReÕ., 60 1980 1284–1335.w x42 Trojanowski, J.Q., Schuck, T., Schmidt, M.L. and Lee, M.V.-Y.,

Distribution of tau proteins in the normal human central and periph-Ž .eral nervous system, J. Histochem. Cytochem., 37 1989 209–215.

w x43 van Leeuwen, J.P., Birkenhager, J.C., van den Bend, G.J., Buurman,C.J., Staal, A., Bos, M.P. and Pols, H.A., Evidence for the func-

Ž .tional involvement of PKC in the action of 1,25 OH D in bone, J.2 3Ž .Biol. Chem., 267 1992 12562–12569.

w x44 Wasserman, R.H., Chandler, J.S., Meyer, S.A., Smith, C.A., Brindak,M.E., Fullmer, C.S., Penniston, J.T. and Kumar, R., Intestinalcalcium transport and calcium extrusion processes at the basolateral

Ž .membrane, J. Nutr., 122 1992 662–671.w x45 Wasserman, R.H., Smith, C.A., Brindak, M.E., de Talamoni, N.,

Fullmer, C.S., Penniston, J.T. and Kumar, R., Vitamin D deficien-cies increase the plasma membrane calcium pump of chicken intes-

Ž .tine, Gastroenterology, 102 1992 886–894.w x46 Wion, D., MacGrogan, D., Neveu, I., Jehan, F., Houlgatte, R. and

Brachet, P., 1,25-Dihydroxyvitamin D is a potent inducer of nerve3Ž .growth factor synthesis, J. Neurosci. Res., 28 1991 110–114.

w x47 Zheng, M. and Heinrich, G., Structural and functional analysis of thepromoter region of the nerve growth factor gene, Mol. Brain Res., 3Ž .1988 133–140.