Corticotropin-releasing hormone induces proliferation and TNF-α release in cultured rat microglia...

Corticotropin-releasing hormone induces proliferation

and TNF-a release in cultured rat microglia via

MAP kinase signalling pathways

Wei Wang, Ping Ji and Kimberly E. Dow

Department of Pediatrics, Queen’s University, Kingston, Ontario, Canada

Abstract

We have previously demonstrated that corticotropin-releasing

hormone (CRH) receptor 1 (CRH-R1) is functionally

expressed in rat microglia. In the present study, we show that

CRH, acting on CRH-R1, promoted cell proliferation and

tumour necrosis factor-a (TNF-a) release in cultured rat

microglia. Exogenous CRH resulted in an increase in BrdU

incorporation compared with control cells, which was

observed in a range of concentrations of CRH between 10 and

500 nM, with a maximal response at 50 nM. The effect of CRH

on BrdU incorporation was inhibited by a CRH antagonist

astressin but not by a cAMP-dependent protein kinase inhib-

itor H89. Exposure of microglial cells to CRH resulted in a

transient and rapid increase in TNF-a release in a dose–

dependent manner. In the presence of astressin, the effects of

CRH on TNF-a release were attenuated. CRH effects on TNF-a

release were also inhibited by specific inhibitors of MEK, the

upstream kinase of the extracellular signal-regulated protein

kinase (ERK) (PD98059) or p38 mitogen-activated protein

kinase (SB203580), but not by H89. Furthermore, CRH induced

rapid phosphorylation of ERK and p38 kinases. Astressin,

PD98059, and SB230580 were able to inhibit CRH-induced

kinase phosphorylation. These results suggest that CRH

induces cell proliferation and TNF-a release in cultured micro-

glia via MAP kinase signalling pathways, thereby providing

insight into the interactions between CRH and inflammatory

mediators.

Keywords: cytokine, extracellular signal-regulated protein

kinase, glia, p38, receptor, signal transduction.

J. Neurochem. (2003) 84, 189–195.

Corticotropin releasing hormone (CRH), a 41-amino-acid

neuropeptide, plays a central regulatory role as a key

mediator of the hypothalamic-pituitary-adrenal system

response to stress (Vale et al. 1981; Owens and Nemeroff

1991; De Souza 1995). CRH elicits its biological effects by

binding to two G protein-coupled membrane receptors

(CRH-R1 and CRH-R2) with signal transduction being

mediated via several intracellular signalling pathways such as

cAMP, protein kinase C, and mitogen-activated protein

kinases (MAPK) (Chen et al. 1993; Lovenberg et al. 1995;

Perrin et al. 1995; Kostich et al. 1998; Li et al. 1998; Cibelli

et al. 2001). CRH receptors are widely distributed in the

central nervous system (CNS) in general concordance with

the distribution of CRH pathways (De Souza 1995). Recent

studies have also indicated a broad range of distribution of

CRH receptors in the immune, cardiovascular and reproduc-

tive systems, suggesting that the receptors may mediate many

diverse functions (Singh and Fudenberg 1988; Audhya et al.

1991; Clifton et al. 1995; Perrin et al. 1995). In addition to

its effects on hormone regulation, CRH has been implicated

in pathological and pathophysiological responses in various

neurodegenerative disorders, including brain traumatic,

ischemic and excitotoxic injuries. These neurodegenerative

disorders are accompanied by an increase in brain CRH and

its receptors (Wong et al. 1995; Greenwood et al. 1997; Roe

et al. 1998; Wang et al. 2000). Several lines of evidence

suggest that CRH contributes to neuronal loss in vitro and

in vivo while some studies show a protective role of CRH.

Received August 2, 2002; revised manuscript received October 22,

2002; accepted October 23, 2002.

Address correspondence and reprint requests to Dr Kimberly E. Dow,

Doran 3, Room 6–303, Apps Medical Research Center, Kingston Gen-

eral Hospital, Kingston, Ontario, Canada K7L 2V7, Canada.

E-mail: [email protected]

Abbreviations used: CREB, cAMP response element-binding protein;

CRH, corticotropin-releasing hormone; CRH-R1, corticotropin-releasing

hormone receptor 1; ERK, extracellular signal-regulated protein kinase;

GFAP, glial fibrillary acidic protein; LPS, lipopolysaccharide; MAPK,

mitogen-activated protein kinases; PKA, cAMP-dependent protein kin-

ase; TNF-a, tumour necrosis factor-a.

Journal of Neurochemistry, 2003, 84, 189–195

� 2003 International Society for Neurochemistry, Journal of Neurochemistry, 84, 189–195 189

(Strijbos et al. 1994; Maecker et al. 1997; Roe et al. 1998;

Lezoualc’h et al. 2000; Brunson et al. 2001; Pedersen et al.

2001). The molecular mechanisms of CRH effects and the

functional importance of CRH receptors during these stress-

related conditions are largely unknown. Some studies have

shown that increased CRH following brain insults plays an

important role in immunomodulation in either an autocrine or

paracrine manner at the level of the CNS (Karalis et al. 1991;

Vamvakopoulos and Chrousos 1994; Chrousos 1995; Der-

mitzaki et al. 2002).

Microglia, brain resident macrophages, play an important

role in immunoregulation in the CNS by interacting with

neurons, astrocytes, and other glial cells (Kreutzberg 1996;

Giulian 1987). Activated microglia are known to have both

restorative and cytotoxic capabilities by releasing a number

of inflammatory mediators such as nitric oxide, interleukines,

and cytokines (Merrill and Jonakait 1995; Kreutzberg 1996).

These inflammatory mediators, e.g. a potential neurotoxic

cytokine tumour necrosis factor-a (TNF-a) induced by brain

insult or by lipopolysaccharide (LPS), participate in regula-

ting inflammation, immune responses, neurotransmission,

and neural cell death/survival. Thus, microglia play a key

role in mediating CNS tissue damage, both in the early stage

of inflammation and the later stage of tissue repair.

Recently we have shown that functional CRH-R1 is

expressed in rat brain microglia (Wang et al. 2002). There-

fore, CRH may regulate microglial functions such as

inflammatory mediator release, by activation of the CRH

receptor in microglia. To further investigate the physiological

significance of such a receptor in microglia, we examined

whether exogenous administration of CRH regulates cell

proliferation and TNF-a release in primary cultures of rat

brain microglia. Because the release of inflammatory medi-

ators is believed to be mediated through signalling pathways

involving MAPKs, such as extracellular signal-related kin-

ases (ERK) and p38 kinase (Robinson and Cobb 1997), we

also examined effects of CRH on activation of ERK and p38

kinases and effects of inhibitors of ERK and p38 kinases on

CRH-regulated TNF-a release.

Materials and methods

Reagents

Peptide analogues (human/rat CRH and astressin) were purchased

from Bachem (Torrance, CA, USA). MEK1/2 inhibitor PD98059

and p38 kinase inhibitor SB203580 were obtained from Calbiochem

(San Diego, CA, USA). The peptide stock solution was prepared by

dissolving CRH in 0.01 N HCl/0.1% BSA in PBS and stored at

) 20�C. Astressin was dissolved in 0.01 N HCl/0.1% BSA in

distilled water and stored at ) 20�C. BrdU cell proliferation enzyme-

linked immunosorbent assay (ELISA) kit was purchased from

Boehringer Mannheim (Mannheim, Germany). Rat TNF-a ELISA

kit was purchased from Biosource International Inc. (Camarillo, CA,

USA). Anti-phospho-ERK (p44/p42) and anti-total ERK (p44/p42)

polyclonal antibodies were obtained from Cell Signaling (Beverly,

MA, USA) as were anti-phospho-p38 MAP kinase and anti-total

p38 MAP kinase antibodies. All other chemicals and reagents were

obtained from Sigma (St Louis, MO, USA).

Microglia cultures

Cultures of microglia were established based on the differential

adherence of cells harvested from embryonic rat cortex as described

previously (Wang et al. 2002). Briefly, mixed cell cultures were

established from E19 rat cerebral cortex. Cortices were dissociated

by passing through a 70-mm Nitex mesh and plated in culture flasks.

Cultures were fed every 4 days with Dulbecco’s modified Eagle’s

medium (DMEM)/F12 containing 10% foetal calf serum (FCS). On

day 12, the cultures were shaken for 10 min. The suspended cells

were plated on uncoated dishes or plates and incubated for 1 h at

37�C. The medium containing suspended cells was discarded and

adherent cells were fed with DMEM containing 10% FCS. Cultures

with cells positive for the astrocyte marker glial fibrillary acidic

protein (GFAP) or for the oligodendrocyte marker galactocerebro-

side were discarded (Wang et al. 2002).

Proliferation assay

Microglia were plated at 5 · 104 cells/well in 96-well plates and

incubated overnight in DMEM containing 10% FCS. Cultured

medium was changed to DMEM containing 0.1% FCS for a further

24 h. Cells were then treated with indicated concentrations of CRH

for 48 h. After exposure to CRH, cultures were analyzed for cell

proliferation by measuring BrdU incorporation using the BrdU cell

proliferation ELISA kit (Boehringer Mannheim) according to the

manufacturer’s instructions. Briefly, 10 lM BrdU was added to

culture medium 6 h before the end of CRH treatment. Then, culture

medium was removed and cells were fixed for 20 min at room

temperature (20�C). Anti-BrdU-antibody was added to wells and

incubated for 60 min and the wells were washed three times with

washing solution. The immune complexes were detected by the

subsequent substrate reaction and reaction products were quantified

by measuring the absorbance at 450 nm. The developed colour and

thereby the absorbance values directly correlated to the amount of

DNA synthesis and the number of proliferating cells.

Measurement of TNF-a release

Microglia were plated into 24-well plates at 2 · 105 cells/well and

incubated overnight. Cells were then treated with various concen-

trations of CRH for different times. TNF-a released into culture

media was measured using rat TNF-a ELISA kits (Biosource)

according to the manufacturer’s instructions. The final concentra-

tions of TNF-a were calculated by converting the optical density

readings using standard curves.

Measurement of ERK and p38 kinase phosphorylations

Western blots were performed for the analysis of ERK and p38

kinase phosphorylation. Briefly, cultured microglia were collected

and lysed in sample buffer and sonicated for 10–15 s. The samples

(80 lg of protein) were boiled for 5 min and electrophoresed on a

10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis

(SDS–PAGE). After electrophoresis, gels were blotted to nitrocel-

lulose membranes. Following blocking with 5% non-fat milk, the

membranes were washed and incubated with primary antibodies

190 W. Wang et al.

� 2003 International Society for Neurochemistry, Journal of Neurochemistry, 84, 189–195

(phospho-p44/p42 and phospho-p38 kinase rabbit polyclonal anti-

bodies at a final concentration of 1 : 1000) overnight at 4�C.

Subsequently, the membranes were washed again and incubated

with HRP-conjugated secondary antibody (1 : 2000) and HRP-

conjugated antibiotin antibody (1 : 1000) for 1 h at room tempera-

ture. The bands were visualized by an enhanced chemiluminescence

detection system (Amersham Pharmacia Biotech, Piscataway, NJ,

USA). To normalize the results, membranes were stripped and

reprobed with antibodies specific for total p44/42 or p38 kinases,

respectively. Protein concentrations of samples were determined by

the method of Bradford using BSA as a standard (Bio-Rad

Laboratories, Hercules, CA, USA).

Statistical analysis

Differences between groups were compared by student’s t-test and

analysis of variance or by Kruskal–Wallis one-way analysis of

variance on ranks using SigmaStat software.

Results

Effect of CRH on proliferation of microglia

To investigate a possible role for CRH in microglia

proliferation, BrdU incorporation was measured as an index

of microglial proliferation. Microglia in DMEM containing

0.1% FCS were treated with indicated concentrations of

CRH, the CRH receptor antagonist astressin, or a combina-

tion of CRH and astressin. As shown in Fig. 1, CRH treated

cells showed an increase to �160% of the control levels in

BrdU incorporation over 48 h in culture. The CRH-induced

increase in BrdU incorporation was dose–dependent in the

concentration range of 10–200 nM and the microglial

response to CRH reached a plateau at 50 nM (Fig. 1). When

microglia were incubated in medium containing 10% serum,

CRH had no effect (data not shown). To study the specificity

of the stimulating effect of CRH on microglial proliferation,

cells were treated with CRH in the presence or absence of the

CRH receptor antagonist astressin. The effect of CRH on

BrdU incorporation was inhibited by 200 nM astressin

(Fig. 2a). Astressin alone at 200 nM did not alter the basal

level of BrdU incorporation.

As activation of CRH receptors is classically associated

with activation of the cAMP-dependent protein kinase (PKA)

pathway, cells were treated with the PKA inhibitor H89 at

30 lM for 1 h before addition of CRH. The effect of CRH on

BrdU incorporation was not attenuated by the presence of

H89 (Fig. 2b).

CRH and its related peptides have been shown to activate

MAP kinase signal transduction pathways and phosphoryla-

tion of the transcription factor calcium/cAMP response

element-binding protein (CREB) in several cell types (Li

et al. 1998; Rossant et al. 1999; Cibelli et al. 2001). The

regulation of cell proliferation is correlated with the activa-

tion of MAP kinases, especially the ERK subgroup (Peletch

and Sanghera 1992; Tournier et al. 1994). The role of MAP

kinase in CRH-induced microglial proliferation was inves-

tigated by using an inhibitor of MEK, an upstream kinase of

ERK1/2, PD98059. This inhibitor at a concentration of

50 lM significantly inhibited CRH-induced proliferation

Fig. 1 Effect of CRH on cell proliferation in microglia. Microglia were

incubated with indicated concentrations of CRH in DMEM containing

0.1% FCS for 48 h. Cell proliferation was measured by BrdU incor-

poration ELISA. Results are taken from three independent cultures

and experiments. Data are mean ± SD. *p < 0.05 versus control.

Fig. 2 Effect of astressin (a), H89 or PD98059 (b) on CRH-induced

proliferation. Microglia were incubated with the CRH antagonist

astressin (AS, 200 nM), PKA inhibitor H89 (30 lM) or ERK1/2 inhibitor

PD98059 (PD, 50 lM) for 1 h prior to addition of 100 nM of CRH. Cell

proliferation was determined 48 h following CRH treatment. Results

are taken from three independent cultures and experiments. Data are

mean ± SD. *p < 0.05 versus control, **p < 0.05 versus CRH.

CRH induces TNF-a release in microglia 191


when incubated with cells for 1 h prior to the addition of

CRH (Fig. 2b).

CRH induces TNF-a release in microglia

To study the effect of CRH on TNF-a release in cultured rat

microglia, microglial cells were treated with the indicated

concentrations of CRH for different times and TNF-a release

into the culture medium was determined. Basal levels of

TNF-a in untreated microglia were low and CRH treatment

induced an increased release of TNF-a compared with

control cells (Fig. 3a). A dose–dependent increase in TNF-arelease by CRH was seen up to 250 nM, with the peak

response at 100 nM (Fig. 3a). The time course of CRH-

induced TNF-a release was determined at 2, 4, 6, 24, and

48 h following CRH treatment. As shown in Fig. 3b,

increased release of TNF-a was detected at 2 h after the

addition of CRH. The peak increase of TNF-a release was at

4 and 6 h and then returned to basal levels at 24 h (Fig. 3b).

The marked decrease of TNF-a release at 24 h could largely

be due to the degradation of TNF-a in the culture medium.

Receptor binding of soluble TNF-a could be a contributing

factor (Pyo et al. 1999; Jana et al. 2002). The effect of CRH

on TNF-a release was attenuated when cells were treated

with 200 nM of astressin for 1 h prior to CRH treatment

(Fig. 4a).

To study whether the cAMP-dependent mechanism is

associated with the effect of CRH on TNF-a release, CRH-

induced TNF-a release was determined in the presence or

absence of H89. H89 at a concentration of 30 lM, when

added to cell cultures for 1 h prior to the addition of CRH,

did not alter CRH-induced TNF-a release (Fig. 4b).

To test whether MAP kinase signalling pathways are

involved in the effect of CRH on TNF-a release, microglial

cells were treated with upstream inhibitor of ERK (PD98059)

or p38 kinase (SB203580) at concentrations of 50 lM for 1 h

prior to the addition of CRH and TNF-a release was

determined. Both PD98059 and SB203580 inhibited the

effect of CRH on TNF-a release (Fig. 5). When both

inhibitors were used simultaneously, TNF-a levels were

further decreased (Fig. 5).

CRH induces activation of ERK and p38 MAP kinases

To further investigate the role of MAP kinase signalling

pathways in CRH-induced TNF-a release, microglial cells

were treated with CRH and the phosphorylation status of

ERK and p38 kinases were analyzed by western blotting

using commercially available antibodies specific for the

active forms of the two kinases. As shown in Fig. 6, CRH

Fig. 4 Effects of astressin (a) or H89 (b) on CRH-induced TNF-a

release. Microglia were pre-treated with the CRH antagonist astressin

(AS, 200 nM) or H89 (30 lM) for 1 h prior to addition of 100 nM of CRH.

TNF-a release was determined at 4 h following CRH treatment.

Results are taken from three independent cultures (n ¼ 18). Data are

mean ± SD. *p < 0.05 versus control, **p < 0.05 versus CRH.

Fig. 3 CRH induced TNF-a release in cultured microglia. Microglia

were treated with (a) indicated concentrations of CRH for 4 h or

treated with (b) 100 nM of CRH for indicated time periods. Amounts of

TNF-a released were determined by TNF-a ELISA. Results are taken

from three independent cultures (n ¼ 18). Data are mean ± SD.

*p < 0.05 versus control.

192 W. Wang et al.


induced a transient phosphorylation of both ERK and p38

kinases. The maximal activation of ERK and p38 kinases by

CRH was observed after 20–30 min (Fig. 6). After being

stripped, the membranes were reblotted with anti-total-p44/

42 or p38 antibodies. Normalizing with non-phosphorylated

forms of p44/42 and p38 kinases confirmed that CRH

specifically altered p42/44 and p38 kinase phosphorylation,

but not expression levels. The effect of CRH on ERK and

p38 phosphorylation was inhibited by 200 nM astressin

(Fig. 6). PD98059 at 50 lM significantly inhibited the CRH

effect on ERK kinase phosphorylation when incubated with

cells for 1 h prior to the addition of CRH (Fig. 6). In a

similar manner, the p38 kinase inhibitor SB203580 at 50 lM

also inhibited CRH-induced phosphorylation of p38 kinase

(Fig. 6). Neither PD98059 nor SB203580 had any effect on

cell viability in a concentration range of 1–100 lM (data not

shown). These results suggest that activation of ERK and p38

MAP kinase pathways by CRH is necessary to trigger TNF-arelease in cultured microglia.

Discussion

We have recently demonstrated the functional expression of

CRH-R1 in cultured microglia of rat brain (Wang et al.

2002). In the present study, we extended our previous

observations on the biological response of microglia to CRH.

Our results suggest that (i) CRH promoted cell proliferation

and induced TNF-a release from cultured microglia via

activation of CRH-R1, (ii) CRH-induced microglial prolif-

eration and TNF-a release were associated with activation of

both ERK and/or p38 MAP kinase signalling pathways, and

(iii) the effects of CRH appeared to be independent of

cAMP-dependent protein kinase pathways.

CRH has been shown to promote or inhibit cell prolifer-

ation via interacting with CRH-R1 in several cell types

(McGillis et al. 1989; Singh 1989; Ha et al. 2000; Carlson

et al. 2001; Mitsuma et al. 2001; Quevedo et al. 2001).

CRH promotes proliferation in developing astrocytes in

cerebellar cultures, an effect which is thought to be mediated

through the cAMP pathway and activation of c-Fos (Ha et al.

2000). In malignant melanoma cells, CRH inhibits prolifer-

ation via activation of CRH-R1 and subsequently altered

intracellular Ca2+ signalling (Carlson et al. 2001). The

results of the present study showed that CRH promoted

microglial proliferation and that the CRH effect was inhibited

by astressin, suggesting involvement of the activated CRH

receptor expressed in microglia. The effect of CRH on

microglial proliferation appears to involve MAP kinase

signalling and is independent of cAMP-PKA pathways. The

mechanisms for the effects of CRH on proliferation are not

yet clear, but may depend on cell types and differential signal

pathways activated.

Fig. 5 Effects of ERK and p38 kinase inhibitors on CRH-induced TNF-a

release. Microglia were incubated with PD98059 (PD, 50 lM) or

SB203580 (SB, 50 lM) for 1 h prior to addition of CRH (100 nM). TNF-a

released was determined at 4 h following CRH treatment. Results are

taken from three independent cultures (n ¼ 18). Data are mean ± SD.

*p < 0.05 versus control, **p < 0.05 versus CRH.

(a) (b)

(c) (d)

Fig. 6 CRH induced phosphorylation of

ERK (a) and p38 kinases (b). Microglia

were treated with 100 nM of CRH for indi-

cated time periods and phosphorylation

status of ERK and p38 kinases were

analyzed by western blot as described in

Materials and methods. Experiments were

done from three independent cultures.

A representative experiment is shown.

Panels (c) and (d) showed the effect of

astressin (AS, 200 nM), PD98059 (PD,

50 lM) or SB203580 (SB, 50 lM) on CRH-

induced ERK and p38 kinases activation.



Studies have suggested that CRH plays a role in immune

function by promoting immune cells to produce inflamma-

tory mediators (Singh and Leu 1990; Angioni et al. 1993).

Our data provide evidence that CRH-induced TNF-a release

from cultured microglia is mediated by activation of the

MAP kinase pathways. It has been demonstrated that CRH

and its related peptides can activate ERK and p38 kinases in

several cell types (Li et al. 1998; Rossant et al. 1999; Cibelli

et al. 2001; Dermitzaki et al. 2002). In the present study, an

increase in tyrosine phosphorylation of ERK and p38 kinases

was detected within 30 min following treatment with CRH.

CRH-induced phosphorylation of ERK and p38 kinases was

inhibited by MEK/ERK (PD98059) and p38 (SB203580)

kinase inhibitors, respectively. The activation of ERK and

p38 was also blocked by the CRH receptor antagonist

astressin. Furthermore, CRH-induced TNF-a release from

microglia was reduced by pre-treatment with both PD98059

and SB203580. While a recent report has shown that CRH

can activate p38 MAPK (Dermitzaki et al. 2002), SB203580

may also inhibit c-Raf, an upstream form of the p44/42 MAP

kinase (Hall-Jackson et al. 1999). Thus, SB203580 may

have its inhibitory effect by acting through p44/42 MAP

kinase. Further studies are required to provide direct

evidence for the involvement of p38 in CRH-induced cell

proliferation and TNF-a release. While we have recently

shown that CRH increases cAMP levels in cultured microglia

(Wang et al. 2002), modulation of cell proliferation and

TNF-a release by CRH may not require the activation of the

cAMP-PKA pathway. Thus the diverse functions of CRH in

the brain and peripheral tissues may be due in part to the

selective activation of these two downstream signal path-

ways.

The immune and neuroendocrine systems are two essential

physiological components linked by reciprocal regulatory

interactions. CRH has been shown to modulate inflammatory

responses by regulating the pro- or anti-inflammatory

mediators at the level of the CNS (Sapolsky et al. 1987;

Karalis et al. 1991; Angioni et al. 1993; Vamvakopoulos and

Chrousos 1994; Chrousos 1995; Paez Pereda et al. 1995;

Linthorst et al. 1997). In light of microglial function in the

CNS, the present results demonstrate a potentially important

function of CRH in both normal homeostasis and neurode-

generative disease states. Therefore, our findings may

provide insight into basic cellular interactions between

CRH and inflammatory mediators in pathological conditions

within the CNS. The interaction may prove to be a very

important mechanism in responses to stressful challenges

such as acute or chronic brain insults.

Acknowledgements

This work was supported by the Principal’s Developmental Fund of

Queen’s University (380–006 to WW) and the Angada Foundation

(365–983 to WW) and in part by the Heart and Stroke Foundation of

Ontario (NA5024 to KED). We thank Dr G. Zhao for his technical

assistance in microglia cultures.

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Corticotropin-releasing hormone induces proliferation and TNF-α release in cultured rat microglia...

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