Antagonistic effects of dexamethasone and 1,25-dihydroxyvitamin D3 on the synthesis of nerve growth factor

Molecular and Cellular Endocrinology, 78 (1991) Rl-R6 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50

MOLCEL

Rl

02546

Rapid Paper

Antagonistic

effects of dexamethasone and 1,25dihydroxyvitamin on the synthesis of nerve growth factor

Isabelle Neveu ‘, Nelly Barbot 2, Frkd&-ic Jehan ‘, Didier Wion and Philippe Brachet ’

D,

’

’ Institut National de la Sante’ et de la Recherche Midicale, Unite’298, and ’ Service de Midecine C, Centre Hospitalier Rigional Unicersitaire, Angers, France (Received

Key words: Nerve growth

factor;

11 April 1991; accepted

1,25-Dihydroxyvitamin

D,; Dexamethasone;

12 April 1991)

L929 fibroblast

Summary

Dexamethasone is known to decrease the pool of nerve growth factor (NGF) mRNA in various experimental systems. The negative regulatory effect of the glucocorticoid was first observed in mouse fibroblast-like L929 cells, and was subsequently reported to take place in many experimental systems, including in vivo following sciatic nerve injury. Conversely, another steroid hormone, 1,25-dihydroxyvitamin D, (1,25-(OH),D,) was recently reported to promote NGF synthesis in mouse L929 cells. The present work was undertaken to investigate the effect of the concomitant addition of both steroids to L929 cells. Measurements of NGF mRNA and assays of the mature protein secreted by the cells provide evidence that the negative regulation exerted by dexamethasone may be counteracted in a dose-dependent manner by the positive action of 1,25-(OH),D,, and vice versa. Therefore, the expression of the NGF gene can be regulated in a subtle way by the balance between the two steroids. It may be expected on the basis of these observations that in tissues that are responsive to both hormones, administration of 1,25-(OH),D, should be able to reverse the down-regulation of NGF synthesis elicited by glucocorticoids.

Introduction

Nerve growth factor (NGF) is a neurotrophic protein which supports the survival of embryonic, sympathetic and some sensory neurons (Thoenen and Barde, 1980). In adult rodents, the death of sensory neurons which takes place following their axotomy may be prevented or minimized upon ectopic addition of NGF (Rich et al., 1987). Con-

Address for correspondence: Isabelle Neveu, 298, CHRU, F-49033 Angers Cedex 01, France.

INSERM

U

versely, the sprouting of sensory endings is impaired in animals injected with NGF antibodies, a treatment which causes deprivation of the endogenous factor (Diamond et al., 1987). A comparable situation is found in the central nervous system where NGF exerts trophic effects on the ascending cholinergic neurons of the basal forebrain (Ghahn et al., 1983; Mobley et al., 1986; Taniuchi et al., 19861, and protects these neurons from death after axotomy (Hefti, 1986; Williams et al., 1986). These observations explain why NGF was proposed to be of potential interest in two widespread human neuropathies: diabetic neu-

R2

ropathy and Alzheimer’s disease. In diabetic neuropathy, a dysfunction of the autonomic and/or sensory nervous sytem has been described (Thomas and Eliasson, 1975; Brown and Asbury, 1984; Niakan et al., 1986). In addition, evidence has been presented for an alteration of endogenous levels of NGF in streptozotocin-induced diabetic rats (Hellweg and Hartung, 1990). In Alzheimer’s disease, the loss of NGF-responsive cholinergic neurons of the basal forebrain represents one of the most consistent hallmarks of the pathology (Coyle et al., 1983). Therefore, the characterization of factors involved in the regulation of the NGF gene appears of importance in respect to pharmacological~ and possibly etiological aspects of both of these diseases. So far, glucocorticoids represent the only hormones known to decrease the level of NGF synthesis. This effect was first shown in L929 cells where dexamethasone was shown to decrease the relative level of NGF mRNA and, as a consequence, the amount of cell-secreted factor (Wion et al., 1986). This observation, also extended to natural glucocorticoids (Siminoski et al., 1987), was subsequently generalized to various types of experimental systems in vitro (Houlgatte et al., 1989; Lindholm et al., 1990), and in vivo following sciatic nerve injury (Linholm et al., 1990). These results suggest that the decision to undertake glucocorticoid treatment should be carefully evaluated in patients with NGF-sensitive nerve lesions (Lindholm et al., 19901. In this context the characterization of factors able to antagonize the glucocorticoid effect on NGF synthesis could be of considerable value. The recent finding that another steroid, 1,25(OH),D,, acts as a potent inducer of NGF synthesis in L929 cells (Wion et at., 19911 prompted us to investigate whether this hormone could reverse the inhibitory effect exerted by dexamethasone on the expression of the NGF gene. Materials

ELISA plates were obtained from C.M.L. (France) and the chemicals from Sigma Chemicals (U.S.A.). Anti-NGF monoclonal antibodies, coupled or not to fi-galactosidase were purchased from Boehringer (F.R.G.). cDNA probes were labelled by random priming using [ 32P]dCTP and a kit obtained from Amersham (U.K.). Culture conditions. L929 mouse fibroblast-like cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% horse serum. Cells were plated at an initial density of 1000 cells/cm”. After 16 h the medium was replaced by a serum-free medium consisting of DMEM/F12 media (3/l, v/v> supplemented with insulin (5 pg/ml), transferrin (5 pg/mll, and selenium (5 ng/ml) (Wion et al., 1985). Cells continued growing exponentially under such culture conditions. After 2 days the serum-free medium was renewed and the steroid hormones added 12 h later. RNA a~ai~sis and ELISA assay. Cells were collected after 7 h of treatment, and total RNA was extracted by the LiCl/urea method (Auffray and Rougeon, 1980). For RNA analyses, 20 pg samples of glyoxal-treated total RNA were loaded onto an 1.2% agarose gel (Thomas, 1980). After fractionation~ RNAs were transferred onto a nylon membrane by capillary blotting and they were subjected to Northern blot analysis according to standard procedures, using a “‘P-labelled mouse NGF cDNA probe (Scott et al., 1983). All blots were then hybridized with a cDNA encoding the rat amyloid precursor protein (Shivers et al., 19X8>, to verify the homogeneity of the RNA loading. The amounts of NGF secreted by the cells in their culture media were measured after 24 h of treatment, with a double-site ELISA assay, as described previously (Houlgatte et al., 19891, using a monoclonal antibody coupled or not coupled to p-galactosidase. Standard curves were obtained and recoveries assessed using purified mouse 2.5s NGF.

and methods Results

Reagents. 1,25-(OH),D, Drs. R. Imhof and U. Fisher, (Basel, Switzerland). Tissue from Gibco (France), tissue was purchased from Nunc

was a kind gift of Hoffmann-La Roche culture media were culture plastic ware (Denmark), O&well

In a first series of experiments, L929 cells cultured in serum-free medium were concomitantly exposed to a constant, elevated dose of dexamethasone (IO-h M) and to increasing con-

R3

centrations of 1,25iUH),R,. The results of a typical Northern blot analysis are shown in Fig. IA. The relative level of NGF mRNA drops in the

1 2 3 4 5

A

6 7 8

6 16 1112

presence of dexamethasone and these transcripts become bareIy detectable isee lanes 1 and 2). Conversely, increasing doses of 1,25(OH),D, induce a dose-dependent accumulation of NGF mRNA, (lanes 3, 5, 7, 9 and 111. When added together with 10m6 M dexamethasone~ 1,25(OH),D, has no detectable effect at the lowest concentration tested (10-‘2 M; Ianes 41, but at higher concentrations, above lo-“” M and up to 10m8 M, a dose-dependent increase of the pool of NGF transcripts is observed, and their levels become higher than those found in control, untreated cells (lanes 6, 8, 10, 12f. In a reciprocal experiment shown in Fig. 1B, cells stimulated with lo-’ M 1,25-(OH),D, were also exposed to increasing concentrations of dexamethasone. In a11 instances the negative effect exerted on the pool of NGF mRNAs by the glucocorticoid alone (lanes 3, 5, 7 and 9) is overcome when 1,25-(OH),D, is concomitantly added to the ceils (lanes 4, 6, 8 and 10). In order to investigate whether the contrasting effect of dexamethasone and I,25-(OH&D, was accompanied by parallel changes in the amounts of NGF secreted by the cells, the factor released in the different supernatant media was assayed after 24 h of treatment. In control cultures, dexamethasone alone causes a decrease of the level of NGF (Fig. 21, while 1,25-(OH),D, has the opposite effect (Fig. 31, Fig. 1. Effect of dexamethasone and 1,25-(OH),D, cotreatment cm the Levels of NGF mRNA in L929 cells. Amyloid precursor protein (APP) mRNA was used as control for the RNA loading. RNA was extracted from cells treated for 7 h with dexamethasone, or 1,25-tOHI,D,, or both. (AI Lane 1, control untreated cells; lane 2, dexamethasone IO-’ M; lane 3, 1,25-(OH&D, lo-‘* M; lane 4, dexamethasone lO_’ M and 1,25(OH),D, 1OV’” M; lane 5, 1,25-(OH),D, 10-t’ M; lane 6, dexamethasone 10e6 M and 1,25-(OH),D, lo-” M; lane 7. 1,25fOHIzD, 1OW’”M; lane 8, dexamethasone 10mh M and 1,25-(OH),D, IO-“” M; tane 9, I.25(OH),D, IO-” M; lane IO, dexamethasone 10mh M and 1,2S(OH),D, IO-.” M; lane 11, 1,25-fOH),D, lo-’ M; lane 12, dexamethasone 10S6 M and 1,25-(OH),D, 10-s M. (B) Lane 1, control untreated cells; lane 2, l,&(OH),D, 10-s M; lane 3, dexamethasone 1O--6 M; lane 4, 1,25-COH)zD, 10-s M and dexamethasone 10m6 M; Iane 5, dexametfiasone 1W7 M; lane 6, 1,25-(OH&D, 10-s M and dexamethasone lo-’ M; lane 7. dexamethasone 10-s M; lane 8, 1,25-(OH&D, 10. ’ M and dexamethasone 10-s M; lane 9, dexamethasone 10-O M; lane 10, 1,2.5-(OH),D, 10. s M and dexamethasone lo-^’ M.

m m

MINIMUM

CONTROL 12 CONTf?OLfO

9

8

DEXAMETHAQONE

DEXAMETHASONE

( IO-*M 1

MEOIUM

f

11

10

g

8

6

(- log M 1

Fig. 2. Amount of extracellular NGF secreted L929 cells cultured in the absence (control) increasing doses of dexamethasone. Each value mean+f three independent experiments and perfoFmed in triplicate. Bars represent SD. * ferencc with control CP c: 0.01, Student’s

durmg 24 h by or presence of represents the al1 assays were Significant difr-test).

Fig. 4. Amounts of extracellular NGF secreted by cells exposed to 10mh M dexamcthasone in the absence (control) or presence of increasing doses of 1,25-(OH),D,. For the statistical significance, see Legend to Fig. 2

7L

65 43-

cn

21 1

CONTROL

12

11

10

9

CONTROLlO

9

DEXAMEfXASDNE l,ZS_/UH)2

Dg f-log

Mf

Rg. 3. Amounts of entracelluiar NGF secreted m rhe absence icontrol) or presence of increasing concentrations of 1,25(OH),D,. For the statistical significance, see legend to Fig. 2.

8

7 (-log

6 M)

5. Amounts of extracelhrlar NGF secreted by ceils expostd to 10~” M 1,25-fClHfzD, in the absence (control) or presence of increasing doses of dexamethasone. For the statistical significance, see legend to Fig. 2.

R5

The action of lop6 M dexamethasone is counteracted by increasing amounts of 1,25-(OH),D, (Fig. 41, and reciprocally, the positive effect of is attenuated when in10-s M 1,25-(OH),D, creasing doses of the glucocorticoid are added to the cells (Fig. 5). Discussion

Interference between 1,25-(OH&D, and dexamethasone has been reported to occur in several systems, where antagonistic or agonistic effects were observed. This is for instance the case for osteocalcin or collagen synthesis in osteoblasts, or calcitonin release by thyroid C cells (Chen et al., 1986; Lazaretti-Castro et al., 19901. Depending on their dose, the expression of the NGF gene is also modulated by the balance between these steroids since 1,25-(OH),D, can counteract the decrease in NGF synthesis induced by dexamethasone, while the glucocorticoid may depress in turn the stimulatory effect of 1,25-(OH&D,. Both steroids appear to act primarily on some pretranslational step since the levels of cell-secreted NGF were closely correlated to those of the NGF transcripts. The molecular mechanisms controlling the amount of NGF mRNA may be complex. Steroid receptors can influence gene expression at the level of target regulatory sequences (Morrison et al., 1989; Lindholm et al., 1990). However, glucocorticoids may also modulate the cellular activity by influencing the number or activity of receptors for 1,25-(OH),D, (Chen et al., 1983; Karmali et al., 19891, or alternatively by interacting with proteins encoded by proto-oncogenes such as c-fos and c-jtin (Jonat et al., 1990; Lucibello et al., 1990; Schiile et al., 1990; Yang-Yen et al,, 1990). Glucocorticoid receptors are found in a wide variety of cell types. Receptors for 1,25-(OHl,D, may be less widespread, but they have been reported to exist in tissues not previously recognized as target organs for the hormone. These include neuronal tissues such as the brain and sensory ganglia but also cells of the immune system, such as macrophages (Peacock et al., 1982; Prowedini et al., 1983). In the same way, extrarenal production of 1,25-(OH),D, has been demonstrated especially in activated macrophages (Reichel et al., 1987). These cells

have been reported to play a crucial role in the induction of NGF synthesis in the lesioned sciatic nerve (Heumann et al., 19871, while glucocorticoids are among the most potent inhibitors of the macrophagic functions (Snyder and Unanue, 1982). Thus the antagonism between glucocorticoids and 1,25-(OHl,D, on NGF gene expression could participate in a complex regulatory mechanism involving the endocrine, immune and nervous systems.

The authors thank Dr. C. Smith for critical reading of the manuscript. The work was in part supported by the Minis&e de la Recherche et Technologie, action ‘Vieillissement’. References Auffray, C. and Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314. Brown. M.J. and Asbury. A.K. (1984) Ann. Neural. 15, 2-12. Chen. T.L., Cone, CM., Morey-Holton, E. and Feldman. D. (1983) J. Bioi. Chem. 258, 4350-4355. Chen, T.L., Hauschka, P.V., Cabrales, S. and Feldman, D. (1986) Endocrinology 118, 250-259. Coyle, J.T., Price, D.L.. and DeLong, M.R. (1983) Science 219, 1184-1190. Diamond, J., Coughlui, M., Macintyre, L., Holmes, M. and Visheaw, B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, hS966600. Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H. (1983) Dev. Brain Res. 9, 45-50. Hefti, F. (1986) J. Neurosci. 6, 2155-2162. Hellweg, R. and Hartung, H.D. (1990) J. Neurosci. Res. 26, 258-267. Heumann, R., Lindholm, I)., Bandtlow, C., Meyer, M.. Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8735-8739. Houlgatte, R., Wion, D. and Brachet, P. (1989) Dev. Brain Res. 47, 171-179. Jonat, C., Rahmsdorf, H.J., Kun Koo, P., Cato, A.C.B., Gebel, S., Ponta, I-l. and Herrlich, P. (1990) Cell 62, 1189-1204. Karmali, R., Farrow, S., Hewison, M., Baker, S. and G’Riordan, J.L.H. (1989) J. Endocrinol. 123, 137-142. Lazaretti-Castro, M., Graver, A., Rave, F. and Ziegler, R. (1990) Mol. Cell. Endocrinol. 71, 13-18. Lindholm. D., Hengerer, B., Heumann, R., Carol& P. and Thoenen, H. (1990) Eur. J Neurosci. 2, 795-801. Lucibello, F.C., Slater, E.P.. Jooss, K.V., Beato. M. and Miiller, R. (1990) EMBO J. 9, 2&27-2834. Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Gemski, J., Buchman, K. and Johnston, M.V. (1986) Mol. Brain Res. 1, 53-62.

R6 Morrison, N.A., Shine, J., Fragonas, J.-C., Verkest, V., McMenemy, M.L. and Eisman, J.A. (1989) Science 246, 1158-1161. Niakan, E., Harati, and Comstock, J. (1986) Metabolism 35, 224-234. Peacock, M., Jones, S., Clumens, T.L., Amento, E.P., Kurnick, J.T., Krane, S.M. and Holick, M.F. (1982) in Vitamin D: Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism (Norman, A.W., Scharfer, K., Herrath, D.V. and Grigoleit, H.G., eds.), p. 8385, Walter De Gruyter, Berlin. Provvedini, D.M., Tsoukas, C.D., Deftos, L.J. and Manolagas, S.C. (1983) Science 221, 1181-1183. Reichel, H., Koeffler, H.P. and Norman, A.W. (1987) J. Biol. Chem. 262, 10931-10937. Rich, K.M., Luszczynski, J.R., Osborne, P.A. and Johnson, E.M. (1987) J. Neurocytol. 16, 261-268. Schiile, R., Rangarajan, P., Kliewer, S., Ransone, L.J., Bolado, J., Yang, N., Verma, I.M. and Evans, R.M. (1990) Cell 62, 1217-1226. Scott, J., Selby, M., Urdea, M., Quiroga, M., Bell, G.I. and Rutter, W.J. (1983) Nature 302, 5388540. Shivers, B.D., Hilbich, C., M&have, G., Salbaum, M., Beyreuther, K. and Seeburg, P.H. (1988) EMBO J. 7, 1365-1370.

Siminoski, K., Rennert, P.D., Murphy, R.A. and Heinrich, G. (1987) Endocrinology 121, 1432-1437. Snyder, D.S. and Unanue, E.R. (1982) J. Immunol. 129, 1803-1805. Taniuchi, M., Schweitzer, J.B. and Johnson, E.M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1950-1954. Thoenen, H. and Barde, Y.A. (1980) Physiol. Rev. 60, 12841335. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 52015205. Thomas, P.K. and Eliasson, S.G. (1975) in Peripheral Neuropathy (Dyck, P.J., Thomas, P.K. and Lambert, eds.), pp. 956-987, Saunders, Philadelphia, PA. Williams, R.L., Varon, S., Peterson, G.M., Wictorni, K., Fischer, W., Bjorklund, A. and Gage, F.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9231-9235. Wion, D., Barrand, P., Dicou, E., Scott, J. and Brachet, P. (1985) FEBS Lett. 189, 37-41. Wion, D., Houlgatte, R. and Brachet, P. (1986) Exp. Cell Res. 162, 562-565. Wion, D., MacGrogan, D., Neveu, I., Jehan, F., Houlgatte, R. and Brachet, P. (1991) J. Neurosci. Res. 28, 110-114. Yang-Yen, H.F., Chambard, J.C., Sun, Y.L., Smeal, T., Schmidt, T.J., Drovin, J. and Karin, M. (1990) Cell 62, 1205-1215.