The role of connexin43-gap junction in rat spinal
astrocytes for the regulation of neuropathic pain
（神経因性疼痛に対する脊髄アストロサイト
connexin43-gap junction の役割に関する研究）

博士論文
2014

広島大学大学院	
  医歯薬学総合研究科	
  薬学専攻	 薬効解析科学講座

平成 23 年度 10 月入学	
  	
  張	
  芳芳
	
  	
  	
 

主指導教員	
  	
  	
 

	
  仲田	
  義啓

	
  	
  	
  	
  副指導教員	
  	
  	
  	
  	
  太田	
  茂
	
  	
  	
  	
 

副指導教員	
  	
  	
  	
  	
  森岡	
  徳光
1

TABLE OF CONTENTS
Abstract (JAPANESE)
Chapter 1. The down-regulation of connexin 43 expressions 	 in spinal astrocytes contributes to the maintenance ofhypersensitivity of neuropathic pain in mice with partial
sciatic nerve ligation
1.1. Abstract3

1.2. Introduction

4

1.3. Materials and Methods

6

1.4. Results

9

1.5. Discussion

13

Chapter 2. Spinal astrocytes stimulated by tumor necrosis
factor-α (TNF-α) and/or interferon-γ (IFN-γ) attenuates
connexin 43-gap junction
2.1.

Abstract

18

2.2.

Introduction

19

2.3.

Materials and Methods

21

2.4.

Results25

2.5.

Discussion

29

Summary35

References

36

Acknowledgements (JAPANESE)

54

2

Chapter 1
The down-regulation of connexin 43 expressionsin spinal astrocytes contributes to themaintenance of hypersensitivity of neuropathic pain
in mice with partial sciatic nerve ligation

1.1.

Abstract

Spinal astrocytes are likely to have key roles in maintaining the chronic pain
state. One characteristic feature of astrocytes is their high expression levels ofconnexin 43 (Cx43), a protein that forms gap junction channel, which facilitate
direct intercellular communication. Several recent studies have indicated that thechange of Cx43 expression level in astrocytes might be associated with theinduction of various kinds of neuronal disorder such as multiple sclerosis, epilepsy
or chronic pain, however, it is yet unknown which kinds of endogenous molecule
could be involved in the change of Cx43 expression, and the mechanism related tothe induction of neuropathic pain after the change of Cx43 expression. The
current study examined the relationship between the change of Cx43 expression
and the regulation of nociceptive responses under neuropathic pain. It was foundthat the expression of Cx43 in spinal dorsal horn in partial sciatic nerve ligation(PSNL) model was significantly decreased during the maintenance phase, andthis response was mediated by tumor necrosis factor-α (TNF-α) dependent
pathway. Furthermore, the decrease of Cx43 expression could be related in the
induction of mechanical hypersensitivity. Most importantly, it is possible that theenhancement of glutamatergic transmission related in the down-regulation of
GLT-1 expression might contribute to the mechanical hypersensitivity evoked by
the decrease of Cx43 expression.
3

1.2.

Introduction

Emerging evidence indicates that the enhancement of sensitivity of nociceptive
primary afferent neurons and hypersensitivity of nociceptive dorsal horn neurons
in spinal cord are associated with pathological chronic pain1,2. Recent studies havedemonstrated that spinal glial cells are involved in the initiation of these
responses, and crucial players in the development and maintenance of chronic
pain related in neuropathic or inflammatory disorders3-5. Especially, activatedspinal astrocytes could contribute to the long-lasting of pain hypersensitivity andthe reduction of astroglial activity by intrathecal (i.t.) treatment with fluorocitrate,
an inhibitor of astrocytic metabolism, significantly improve abnormal pain
responses in rodent neuropathic pain models6. There are a number of reports
regarding change of astrocytic function under neuropathic pain; activation of MAP
kinases, increase of proinflammatory cytokines and chemokines production, or
down-regulation of excitatory amino acid transporters (EAATs, Na+-dependentglutamate transporters)7-9. Furthermore, it has been demonstrated that these
responses could contribute to the maintenance of neuropathic pain state.
It has been recently shown that astrocytes intercellular communication throughgap junctions has a crucial role in not only the regulation of astroglial function but
also the proper maintenance of neuronal network10. Gap junctions have important
property for molecular exchange and informational communication by passing
several signal molecules such as glutamate, ATP, and some second messengers,
buffering extracellular Na+ or K+ spatially, or supplying energy sources between
neighboring cells11,12. Gap junctions are formed by two connexons expressed in
distinct cell, which are consisted of a hexamer of connexins (Cxs) protein. It is
known that 11 of Cxs are detected in mammalian central nervous system, andeach cell type possess a distinct set of Cxs. In spinal cord, Cx43 is preferentially4and mainly expressed in astrocytes13. Several studies have indicated thatastrocytic Cx43 plays an essential role in the modulation of synaptic transductionin central nervous system. In addition, the change of Cx43 expression level inastrocytes might be associated with the induction of various kinds of neuronal
disorder. Especially, it has been demonstrated that the decrease of Cx43 protein
expression in astrocytes could enhance the neuronal excitability, and this
response might contribute to the initiation of neuroinflammation such as multiple
sclerosis14. Emerging evidences have suggested that the up-regulation of Cx43 inspinal cord was induced after inflammatory-related pain models, and the
reduction of Cx43 activity by intrathecal delivery of specific siRNA for Cx43 or gap
junction inhibitor carbenoxolone could improve the decrease of nociceptive
threshold15,16. In contrast, Ohara et al. have also indicated that silencing of Cx43
in the trigeminal ganglion of naïve rat administered by siRNA increased
mechanical sensitivity. Thus, relationship between the expression level of Cx43
and the change of nociceptive responses is controversial17. In addition, it is yet
unknown, which kinds of endogenous molecule could be involved in the change ofCx43 expression, and the mechanism related to the induction of neuropathic pain
after the change of Cx43 expression.
To understand the relationship between the change of Cx43 expression and theregulation of nociceptive responses under neuropathic pain, the current study
utilized partial sciatic nerve ligation (PSNL) model, which mimics some of the
major features observed in clinical neuropathic pain. It was found that theexpression of Cx43 in spinal dorsal horn following PSNL was significantly
decreased during the maintenance phase, but not the induction phase of
neuropathic pain, and this response was mediated by tumor necrosis factor-α(TNF-α) dependent pathway. Furthermore, the decrease of Cx43 expression could5be related in the induction of mechanical hypersensitivity. In addition, it ispossible that the enhancement of glutamatergic transmission related in the
down-regulation of GLT-1 expression might contribute to the mechanicalhypersensitivity evoked by the decrease of Cx43 expression.

1.3. Materials and Methods
Animals
Male ddy mice, 5 weeks of age, were used for the experiments. Mice weremaintained at a room temperature of 22±2 0C with a 12h light/dark cycle (light on
at 8:00 AM), and given access to food and water available ad libitum during theexperimental period. All experiments by using animals were conducted inaccordance with the “Guidelines for the Care and Use of Laboratory Animals”
established by Hiroshima University, and the procedures of all animal
experiments were approved by the Committee of Research Facilities forLaboratory Animal Science of Hiroshima University.
Construction of partial sciatic nerve ligation model
After anesthetization with sodium pentobarbital (50 mg/kg, i.p.), a tight ligationof approximately one-third or one-half of the diameters of the left sciatic nerve(ipsilateral) was performed with 8-0 silk sutures as described previously18. In
sham-operated mice, the sciatic nerve was exposed without ligation.
Knockdown of Cx43 in the spinal cord
Knockdown of Cx43 was performed by using the hemagglutinating virus of theJapan (HVJ) envelop vector system (HVJ Envelop Vector kit GenomONE-Si,
Ishihara Sangyo Kaisya, Ltd., Osaka, Japan). This vector was widely used for invivo small interfering RNA (siRNA) transfers. The siRNA targeting mouse Cx43
(siGENOME SMARTpool, mouse GJA1, Thermo) or the non-targeting siRNA6

(siGENOME Non-targeting siRNA pool #2, Thermo) were incorporated into the
HVJ envelop vector according to the manufacture’s protocol. In brief, after mixing
HVJ envelop vector with enclosing factor, the mixture was centrifuged at 10,000 g,
4 0C, 10 min, and the pellet was suspended by the stock solution of each siRNA (10µM). Then, this solution was diluted to the concentration used in the current
study with sterile saline. Diluted siRNA solution was injected into thesubarachnoid space between the L5 and L6 vertebrate (i.t.) of mice.
Measurement of hind paws sensitivity to tactile stimulation and drugs
administration
The procedure of measurement of hind paw sensitivity of ipsilateral paw totactile stimulation was described in previous report. In brief, after surgery for 3, 7,
14, or 21 days, the threshold to tactile stimuli by von Frey monofilaments wasmeasured. About drug treatments, etanercept (TNF-α blocker) were i.t.
administrated four times; immediately after sciatic nerve injury, on the day 2, 4,
or 6 after the injury. Then, the mechanical sensitivity or the expressions of Cx43
were measured on the day 14 after surgery. TNF-α (mouse recombinant TNF),
Carbenoxolone (gap junction blocker) or gap27 (selective Cx43 blocker) was i.t.
injected to naïve mice.
The hind paw sensitivity of spinal cord Cx43 knockdown mice were measured
after injection of siRNA for 1st, 2nd, 3rd, 5th and 7th day by von Frey monofilaments.
About drug treatments, MK-801 (N-methyl-D aspartate receptor antagonist),CP96345 (neurokinin 1 receptor antagonist), CNQX (AMPA receptor antagonist)
were i.t. administrated on the day 3 after injected the siRNA.
Western blot
Under anesthesia with ether, mice were decapitated; the lumbar (L4-L6)
segments of ipsilateral side of the spinal dorsal horn were removed. These were7

immediately frozen in liquid nitrogen and stored at -80 0C until use. Spinal
tissues were solubilized in radioimmunoprecipitation assay buffer with inhibitors(100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 µg/ml aprotinin, 20µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor
cocktail 2 (Nacalai Tesque, Kyoto, Japan)). The lysates were centrifuged at 14,000
× g for 10 min at 4℃ and the supernatant was added to Laemli’s buffer and boiled
for 5 min. Equal amounts of protein were separated by 7.5 or 10%
SDS-polyacrylamidegelelectrophoresis

andblottedonto

nitrocellulose

membranes. Non-specific binding was reduced with blocking buffer, and themembranes were subsequently incubated with a purified polyclonal antibody
against rat Cx43, or GLT-1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA)or monoclonal antibody against β-actin (1:10,000; Sigma Chemical Co., St. Louis,
MO, USA) overnight at 4 0C. After being washed, the membranes were incubatedwith a horseradish peroxidase-conjugated secondary antibody (Santa Cruz
Biotechnology) for 1 hour at room temperature. Then, membranes were rinsed
and incubated with Luminescence reagent (Thermo Fisher Scientific, Rockford, IL,
USA). Finally, the membranes were exposed to X-ray film. For quantification of
signals, the densities of specific bands were measured with Science Lab Image
Gauge (Fuji Film, Tokyo, Japan).
Reagents
Recombinant mouse TNF-α was obtained from PeproTech Inc. (Rocky Hill,
USA) and etanercept was obtained from takeda pharmaceutical co ltd (Osaka,japan). MK801 was purchased from Tocris Bioscience (Minneapolis, MN, USA),
CNQX, CP96345 and Carbenoxolone were purchased from Sigma Chemical, Co.
(St. Louis, MO, USA). Cabapentin was obtained from Cayman Chemical (A. G.
8

Scientific, USA)
Statistical analysis
Data are expressed as the mean±SE of at least three independent
determinations. Differences between means for mechanical hypersensitivity after
PSNL or the knockdown of Cx43 were determined using a one-way analysis ofvariance (ANOVA) with a pairwise comparison by the Tukey-Kramer method.
Using student’s t-test performed comparison of Cx43, GLT-1, Cx30 or GLAST
protein level in the spinal dorsal horn. Differences were considered to besignificant when the P value was less than 0.05.

1.3. Results
PSNL

evokesmechanicalhypersensitivity

anddecreases

theexpression of Cx43 protein expression in spinal dorsal horn
It was observed a marked decrease of mechanical threshold of ipsilateral paw totactile stimuli from days 3 to days 21 after PSNL surgery, compared withwithdrawal threshold of corresponding day after sham operation (Fig. 1-1A).
About Cx43 expression, three immunopositive bands were detected at
approximately 39-44 kDa (Fig. 1-1B) by Western blotting using a Cx43 antibody.
“P0” in Fig. 1-1B indicates the non-phosphorylated Cx43, and P1 and P2
correspond to phosphorylated Cx43. In the current study, total Cx43 expression
was derived from the sum of the three bands (P0+P1+P2). The amount of Cx43
levels was normalized to that of β-actin levels, which was used as an internal
control, and presented as a ratio to vehicle treatment. Then, the expression levelof Cx43 protein in spinal dorsal horn was examined on days 3, 7, 14, 21 after
surgery by Western blot. As shown in Fig. 1-1B, levels of Cx43 expression in
ipsilateral dorsal horn was significantly reduced on days 7, 14, and 21, but not9

days 3, after sciatic nerve injury, compared with levels of Cx43 in dorsal hornfrom sham-operated mice.
Knockdown of Cx43 by siRNA in the spinal cord induces themechanical hypersensitivity
To confirm the specific down-regulation of Cx43 in spinal dorsal horn exerting
mechanical hypersensitivity, the effect of knockdown of Cx43 by siRNA transfer
on tactile mechanical threshold was investigated. The significant reduction of
Cx43 expression in spinal dorsal horn was observed on days 2 or 3 after injection
with Cx43 siRNA with HJV envelop vector compared with that treated with
non-targeting siRNA with HJV envelop vector (Fig. 1-2A), and this response wasreturned on days 5. Furthermore, a significant decrease of mechanical thresholdto tactile stimuli was also observed in mice treated with Cx43 siRNA, comparedwith those in mice treated with non-targeting siRNA. These effects were appearedon days 2 after injection with Cx43 siRNA, sustained on days 5, and disappearedon days 7 (Fig. 1-2B). In addition, the effects of a gap junction blocker(carbenoxolone) on the mechanical threshold were examined. As shown in Fig.
1-2C, i.t. treatment with either carbenoxolone (1 or 3 nmol) markedly decreased
the mechanical threshold at 24 h after injection, compared with treatment withvehicle (saline). Thus, these results strongly indicate that the decrease of
Cx43-gap junction could induce mechanical hypersensitivity.
The change of Cx30 expression in spinal dorsal horn after PSNL
treatment
Previous studies have demonstrated that the enhancement of expression of
Cx30, which is another types of Cxs expressed in astrocytes, was induced after the
down-regulation of Cx43 in astrocytes19. Thus, the expression level of Cx30
protein in spinal dorsal horn was examined on days 7, 14 after surgery that Cx43
10

decreased time pint by Western blot. As shown in Fig. 1-3A, the level of Cx30
expressions in ipsilateral dorsal horn was not changed after sciatic nerve injury,compared with levels of Cx30 in dorsal horn from sham-operated mice. In addition,
we also examined the effect of knockdown of Cx43 by siRNA transfer on Cx30
expression in spinal dorsal horn. As shown in Fig. 1-3B, the level of Cx30
expressions in spinal dorsal horn also not changed after treated with Cx43 siRNAwith HJV envelop vector (Fig. 1-3B).
TNF-α dependent pathway is involved in the PSNL-induced Cx43 down
regulation
Several studies have shown that proinflammatory cytokines such as TNF-α
released in the spinal cord after nerve injury, and active the astrocyte theninduced the neuropathic pain20. Thus, the involvement of endogenous TNF-α inthe down-regulation of Cx43 expression after sciatic nerve injury was investigated.
As shown in Fig. 1-4A or B, repeat i.t. treatment with etanercept, a TNF-α blocker,
was significantly reversed the PSNL-induced mechanical hypersensitivity.
Furthermore, it was also found that blockade of TNF-α’s action could significantly
prevent the down-regulation of Cx43 expression in spinal dorsal horn of PSNL
mice (Fig. 1-4C). Treatment of sham-operated mice with etanercept with sameschedule had no effects on the mechanical threshold and Cx43 expression (Fig.
1-4B or C). In addition, single i.t. treatment with recombinant TNF (20 ng) for 24
or 48 hours markedly induced both mechanical hypersensitivity (Fig. 1-4D) andthe down-regulation of Cx43 expression (Fig. 1-4E).
The down-regulation of GLT-1 and subsequent enhancement ofglutamatergic transmission in spinal dorsal horn is involved in the
Cx43-mediated mechanical hypersensitivity
It is speculated that the enhancement of nociceptive transmission at spinal
11

dorsal horn level might be associated with mechanical hypersensitivity triggeredby the down-regulation of Cx43. Therefore, the involvement of glutamate or
substance P, which acts as main neurotransmitters of nociceptive transduction in
dorsal horn, in Cx43 siRNA-induced mechanical hypersensitivity, was examinedby pharmacological approaches. As shown in Fig. 1-5A and B, i.t. treatment with
MK-801 (a NMDA receptor antagonist) or CNQX (an AMPA receptor antagonist)
led

to

a

significant

blockade

of

the

Cx43

siRNA-induced

mechanicalhypersensitivity. In contrast, i.t. treatment with CP96345 (an NK1 receptor
antagonist) did not block the Cx43 siRNA-induced responses (Fig. 1-5C). These
receptor antagonists had no effects on the mechanical threshold of micetransfected with non-targeting siRNA (Fig. 1-5A-C). These observations led us to
speculate the enhancement of glutamatergic transmission at dorsal level under
the situation which spinal Cx43 expression is down regulated. In addition we
investigate the effect of gabapentin that enhancer of glutamate release. As shownin Fig. 1-5D, i.t. treatment with gabapentin did not block the Cx43 siRNA-induced
responses (Fig.1-5D).
Thus, we next investigated that the expression of glutamate transporters inspinal dorsal horn might be mediated by the change of Cx43 expression level. As
shown in Fig. 1-6A, the expression of GLT-1, which is one of glial glutamate
transporters and abundantly expressed in astrocytes, was significantly reduced inspinal dorsal horn on days 7, 14 or 21, but not days 3, after peripheral nerve
injury, compared with those in sham-operated mice. The current observation was
similar with previous reports. Furthermore, it was found by Western blot that theexpression of GLT-1 in spinal dorsal horn was dramatically reduced on days 2, 3,
5 after the intraspinal transfection with Cx43 siRNA, compared with thosetreated with non-targeting siRNA (Fig. 1-6B). This positive relationship between
12

Cx43 and GLT-1 was also confirmed by pharmacological approach. Previous
report has suggested that the prolonged treatment with carbenoxolone reduced
not only the function of gap junction but also the expression of connexin43. In thecurrent study, both the expression of Cx43 in spinal dorsal horn and mechanicalthreshold were markedly reduced after intrathecal administration withcarbenoxolone for 24 hours (Fig. 1-6C). In addition, it was found that theexpression of GLT-1 was also down regulated by the treatment withcarbenoxolone (Fig. 1-6D).
The expression of GLAST, which is another type of glial glutamate transporters,in spinal dorsal horn, was also elucidated. Although the expression of GLAST inspinal dorsal horn was also significantly reduced by the sciatic nerve injury, thatwas not affected by the knockdown of Cx43 by siRNA (Fig. 1-7A or B).

1.5. Discussion
The current study demonstrated that the TNF-α-modulated down-regulationof Cx43 in spinal dorsal horn could contribute to the PSNL-induced mechanicalhypersensitivity during the maintenance phase (7, 14, 21 days), but not theinduction phase (3 days), of neuropathic pain. These results are supported by thefollowing observations. The knockdown of Cx43 by transfection with siRNA intomice spinal dorsal horn led to a significant decrease of mechanical threshold.
Furthermore, it was found that the down-regulation of glutamate transporter
GLT-1 and the subsequent enhancement of glutamatergic transmission in spinal
dorsal horn might be pivotal events in the Cx43-mediated mechanicalhypersensitivity under sciatic nerve injury. These results indicate that
TNF-α-mediated reduction of Cx43 expression in spinal dorsal horn is associatedwith the maintenance of the neuropathic pain state, and modulation of Cx43
13

expression could be a unique therapeutic strategy for relieving neuropathic pain.
It has well demonstrated that astrocytic dysfunction could be a crucial
response observed under neuropathic pain state. Especially, several previous
reports indicated that the excessive activation or gliosis of astrocytes in dorsal
horn were observed during the maintenance phase, but not the induction phase, of
neuropathic pain21,22, suggesting that astrocytes might play an important role in
the formation of persistent pain. The functions of astrocytes are regulated by theactivities of various kinds of receptor, ion channel, enzyme, etc. Among them, theemerging evidences have shown that Cx-gap junction is indispensable for the
maintenance of astrocytic function23-25. Therefore, it is speculated that the changeof Cx43 expression and the subsequent disturbance of gap junction activity might
be attributed to the various types of neuronal disorders including neuropathic
pain. In fact, previous studies have suggested that the increase of Cx43
expression was observed in spinal cord of nerve-injury-induced neuropathic pain
models, and treatment with gap junction blocker carbenoxolone inhibited thenociceptive responses15,16. In the current study, in contrast, the opposite response
for the expression of Cx43 in spinal dorsal horn was observed, and i.t. treatmentof naïve mice with either carbenoxolone markedly decreased the mechanicalsensitivity. In addition, Wu et al. have demonstrated that the reduced astroglialcommunication by the increase of Cx43 phosphorylation was appeared in the
spinal cord of rat after chronic constriction injury of the sciatic nerve26. Moreover,
Ohara et al. have also indicated that silencing of Cx43 in the trigeminal ganglionof naïve rat administered by siRNA increased mechanical sensitivity17. Although
there is discrepancy between the up- or down-regulation of Cx43 expression in
spinal dorsal horn and nociceptive responses under neuropathic pain state, it may
be based on the differences of neuropathic pain models, pain intensity, or
14

experimental

conditions.

Previous

studieshavedemonstrated

that

the

enhancement of expression of Cx30 was induced after the down-regulation or
knockdown of Cx43 in astrocytes19. In addition, astrocytic Cx30 plays animportant function in neuronal transmission in hippocampus11,27. Therefore, theseobservations suggest the possibility that the up-regulation of Cx30 expression in
spinal dorsal horn might be associated with the Cx43-mediated mechanicalhypersensitivity in the PSNL mice. However, this possibility might be excluded,
because the current observation revealed that the expression of Cx30 was notchanged after the sciatic nerve injury. Thus, the reduction of spinal Cx43
expression from basal level might be one of key responses to induce themechanical hypersensitivity under neuropathic pain state.
TNF-α is well known to contribute to the induction of neuropathic pain throughthe activation of astrocytes20. Therefore, we specially focused the role of TNF-α inthe regulation of spinal Cx43 expression in PSNL mice. In the current study, theobservations obtained by pharmacological approaches using the TNF-α related
reagents indicated that the TNF-α might have a crucial role in the reduction of
Cx43 expression in spinal dorsal horn under neuropathic pain state. Previous
reports demonstrated that the high amount of TNF-α is produced from activatedmicroglia in spinal cord of neuropathic pain models20. It was demonstrated that
increased TNF-α could affect synaptic nociceptive transduction via the regulationof various kinds of astrocytic function28. The current observation strongly
suggests the relationship between the potential roles of TNF-α as a nociceptive
modulator and the regulation of Cx43 expression in spinal astrocytes.
Previous studies have indicated that the down-regulation of GLT-1 in spinal
dorsal horn might be involved in the PSNL-induced mechanical hypersensitivity29.
Furthermore, it is well known that the enhancement of glutamatergic
15

transmission in spinal dorsal horn might be pivotal in the nerve-injury-induced
hypersensitivity30.

However,themolecular

mechanism

initiatingthedown-regulation of GLT-1 is unknown until now. The current observations
indicate the positive relationship between Cx43 and GLT-1, but not GLAST, andthe involvement of the down-regulation of GLT-1 expression in the Cx43-mediatedmechanical hypersensitivity by the following reasons. 1) i.t. treatment withantagonists of either NMDA or AMPA receptor, but not gabapentin, which
inhibits the release of glutamate from presynaptic nerve, significantly reversed
the mechanical hypersensitivity induced by the knockdown of Cx43. 2) The
expression of GLT-1 in dorsal horn was observed in not only the PSNL mice but
also the Cx43-knockdown mice. Together, these current results revealed that the
down-regulation of spinal Cx43 would contribute to the increase of glutamate insynaptic cleft by down-regulation of GLT-1, but not enhancement of presynaptic
release, and the following induction of NMDA receptor-dependent glutamatergictransmission in spinal dorsal horn. Then, these responses could affect the nerve
injury-induced mechanical sensitivity. Previous study indicated that theexpression of GLT-1, but not GLAST, is reduced in cultured cortical astrocytes
treated with gap junction blockers or transfected with Cx43 siRNA31.
Furthermore, the enhancement of glutamatergic current by the decrease ofglutamate transporter’s activity was observed in hippocampus slice of
Cx43/30-double knockout mice27. The molecular mechanisms involving thedown-regulation of GLT-1 after the reduction of Cx43 are yet unclear. Althoughfurther investigation is necessary, emerging evidences have indicated that Cx43
could also function to provide regulatory crosstalk with other proteins located oncell membranes or cytoskeletons, beside the formation of gap junction to passseveral molecules between cells32,33. Furthermore, it was demonstrated that16

Cx43-gap junction might be involved in the regulation of other multiple gene
expression. In fact, it is reported that the alteration of various kinds of gene iscaused in Cx43-null astrocytes34. In addition, Ai et al. have suggested that Cx43 isco-localized with β-catenin in cell membranes; the expression of Cx43 might
sequesterβ-catenin

andnegatively

mediatesβ-catenin-dependent

gene

transcription in cardiac myocytes35. The current observation indicated that theblockade of gap junction function by the intrathecal treatment with gap27 did notaffect the expression of GLT-1 in cultured spinal dorsal astrocyte. Therefore, it ispossible that the down regulation of Cx43 expression might affect the activities of
intracellular signal molecules and the following regulation of various gene
expressions.
Here, we have provided new information about the mechanisms of neuropathic
pain. Under neuropathic pain, Cx43 was decreased via TNF and then induced the
GLT-1 down regulation in the spinal dorsal horn. Furthermore, these responses
werecontribute

to

the

mechanicalglutamatergic transmission.

17

hypersensitivity

viaenhancedtheChapter 2
Spinal astrocytes stimulated bytumor necrosis factor-α (TNF-α) and/or interferon-γ (IFN-γ)
attenuates connexin 43-gap junction

2.1. Abstract
Spinal astrocytes have important mechanistic contributions to the initiationand maintenance of various neuropathic diseases such as neuropathic pain.
Under inflammatory conditions, spinal astrocytes are exposed to cytokines suchas tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) and these cytokines
could alter astrocytic function by modulating connexin (Cx43), subunits that formchannels that modulate intercellular communication in astrocytes. The current
study investigated the alteration of Cx43-gap junction in rat primary cultured
spinal astrocytes stimulated with cytokines by real-time PCR and Western
blotting. The transcriptional and translational levels of Cx43 were significantly
but partially reduced 24 and 48 h treatment with either TNF-α or IFN-γ. A
mixture of TNF-α and IFN-γ led to a robust decrease of Cx43 expression,
moreover, a moderate reduction of gap junction intercellular communication(GJIC), which was evaluated by a scrap loading/dye transfer assay. Both thedecrease of Cx43 expression and the reduction in GJIC induced by the mixture of
TNF-α and IFN-γ were prevented by blocking c-jun terminal kinase (JNK), but
not by blocking extracellular signaling molecules ERK and p38 kinase, indicatinga specific role of astrocytic JNK in the response to cytokines. In addition,
treatment with cytokines potently induced the phosphorylation of JNK and c-jun
inatime-dependent

manner.

Furthermore,theinvolvementofubiquitin-proteasome system on the TNF-α and IFN-γ-induced down-regulation of18

Cx43-gap junction in spinal cultured astrocytes also invested. TNF-α and IFN-γ
mixture accelerated Cx43 degradation by used cycloheximide plus assay in rat
primary cultured spinal astrocyte. Moreover, proteasome inhibitors MG132 or
epoxomicin blocked both the decrease of Cx43 expression and gap junction
function. Furthermore, a mixture of TNF-α and IFN-γ let to a significantly
increase of proteasome activity in rat primary cultured spinal astrocytes by 20s
proteasome activity assay, and the increase of proteasome activity ware prevented
by blocking JNK signaling. In addition, mixture of JNK inhibitor and proteasome
inhibitor were not observed additive inhibitory effect on Cx43 degradationinduced by the mixture of TNF-αand IFN-γ. These results indicate thatintercellular communication of astrocytes is significantly disrupted in the
inflammatory state, and stimulation of spinal astrocytes with inflammatory
cytokines led to the significant inhibition of Cx43-GJIC through the activation of
JNK signaling pathway and proteasome pathway.

2.2. Introduction
Astrocytes are one of the major non-neuronal cell types in the central nervous
system (CNS). One characteristic feature of astrocytes is their high expression
levels of connexin 43 (Cx43), a protein that forms two types of channels: gap
junction channels, for direct intercellular communication, and hemichannels, forsampling of the extracellular milieu 36-39. It is known that the change in cell
coupling resulting from tissue injury and pathological conditions is attributed toalterations in connexin expression and the subsequent modulation of gap junction
intercellular communication (GJIC) 36,40. In terms of brain astrocyte function,
while previous finding highlights the crucial role of Cx43 in the normal
physiological state, Cx43 also appears to be crucial in pathological states. For
19example, Pu et al. showed that Cx43 expression level was inversely correlatedwith high-grade astrocytic tumors 41 and Xu et al. showed that astroyctic gap
junction coupling may contribute to epileptogenesis and other neurologicaldeficits in Tuberous Sclerosis Complex 42. The modulation of Cx43 and GJIC in
astrocytes had been shown to play important roles in the pathology of Parkinson's
disease 43. To elucidate mechanisms of neuropathological states and, in turn, todevelop effective therapeutics, understanding the mechanism of communication
between astrocytes could be as crucial as understanding inter neuronalcommunication.
Recently, several studies have reported expression of Cx43 in spinal astrocytes
in pathological states. Connexin 43 in spinal astroyctes has been shown to besignificantly down-regulated in inflamed spinal white matter of mice with anexperimental autoimmune encephalomyelitis compared with healthy controls 14.
Lee et al. demonstrated that Cx43 is up-regulated in the spinal cord after spinal
cord injury 44. Furthermore, prior chapter showed Cx43 of spinal cord astrocyte
was involved the neuropathic pain. While changes in Cx43 expression in thepathological state are clearly evident, the mechanism mediating the changes ineither expression or function of Cx43 in spinal astrocytes remains unclear.
Studies in brain astrocytes have reported that proinflammatory cytokines
released from activated microglia inhibit gap junction activity in brain astrocytes
24,45, Of these cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) were identified as most responsible for disrupting astrocytic GJIC 24.
Furthermore, prior chapter also showed Cx43 of spinal cord astrocyte was down
regulated by TNF in neuropathic pain. Significant release of these particular
cytokines in the spinal cord have also been reported following spinal cord injury
and peripheral tissue injury (e.g. sciatic nerve injury), which also results in20

significant changes in glial function and proliferation 46,47. Whether a similarcascade occurs in spinal astrocytes, involving cytokines, Cx43 and ultimately
GJIC, has yet to be clarified.
The current study examined whether proinflammatory cytokines, such as
TNF-α, IL-1β and interferon-γ (IFN-γ), could cause an alteration of Cx43
expression and a subsequent functional modulation of GJIC in cultured rat spinal
astrocyte. Mitogen-activated protein kinase (MAPK) is well known to be activatedafter stimulation with cytokines in astrocytes and could be important in initiating
the cascade of intracellular events that leads to various neurological disorders
following injury or disease 48,49. Recently, several researches have focused on therole of ubiquitin proteasome system on Cx43 degradation in several cells such as
human corneal fibroblasts or rat liver epithelial cell line IAR20 50,51. The
ubiquitin-proteasome system is widely known for its role in intracellular protein
degradation, that the proteins after labeled with ubiquities were degradation byproteasome which a large multi-subunit protease 52. There are a number ofsignaling molecules that are activated following cytokine stimulation. Thus, thespecific signaling cascade involved in the expression of Cx43 in astrocytes
following cytokine stimulation was also elucidated.

2.3. Materials and Methods
Reagents
Recombinant rat TNF-α and IFN-γ were obtained from Wako (Tokyo, Japan)
and IL-1β from PeproTech (Rocky Hill, NJ, USA). Lucifer yellow CH di-potassium
salt, H89, N-acetylcysteine (NAC) and Thiazolyl Blue Tetrazolium Bromide(MTT) were purchased from Sigma Chemical, Co. (St. Louis, MO, USA). U0126,
SB202190, SP600125, PP1, Epoxomicin and genistein were purchased from Tocris
21

Cookson (Bristol, UK). Gö6983 and AG490 were obtained from Calbiochemical Co.
(La Jolla, CA, USA). MG132 and 20s proteasome activity assay kit were obtained
from Cayman Chemical (A. G. Scientific, USA). Epoxomicin was purchased from
Calbiochemical Co. (La Jolla, CA, USA).
Primary culture of neonatal rat spinal cord astrocytes
Primary spinal astrocytes were prepared from spinal cords of neonatal Wistar
rats (1–2 days old) according to a previously reported method 53. In brief, theisolated spinal cords were minced and then incubated with trypsin and DNase I.
Dissociated cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat-inactivated fetal calf serum andpenicillin/streptomycin (100 U/ml and 100 µg/ml, respectively). Then, cellsuspensions were plated in 75 cm2 tissue culture flasks (7.5~10×106 cells/flask)
precoated with poly-L-lysine (10 µg/ml). Cells were maintained in a 10% CO2
incubator at 37 °C. After 8~15 days, the mixed glial cells were shaken vigorously
and washed with PBS in order to remove other cells such as microglia. Cells werethen replanted onto 35 mm diameter dishes (3.2×105 cells) for use in the following
experiments. The current method yielded a purity of > 95% astrocytes, as
determined by glial fibrillary acidic protein (GFAP) immunoreactivity (Data notshown).
Real-time PCR analysis
According to a previously reported method 54, total RNA derived from astrocytes
was prepared by a method involving acid guanidinium thiocyanate–phenol–
chloroform extraction 55 and used to synthesize cDNA with moloney murine
leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA)and a random hexamer primer. The cDNA synthesized using 1 µg of total RNA ineach sample were subjected to real-time PCR assays with specific primers and22

EXPRESS SYBER® Green ERTM qPCR SuperMix (Invitrogen, Carlsbad, CA, USA).
The sequences of primers were used: Cx43, 5’ - CGTGCAGATGCACTGAA -3(forward); 5’ - CCACTGGATGAGCAGGAA -3 (reverse) andglyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5’ AGCCCAGAACATCATCCCTG -3 (forward); 5’ - CACCACCTTCTTGATGTCATC-3 (reverse). Thereafter, real-time PCR assays were conducted using a DNA
engine real-time PCR detection system CFX96 (Bio-Rad Laboratories, Tokyo,
Japan). A three-step amplification protocol was applied: 3 min at 95 °C followed
by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. RNA quantities oftarget genes were calculated by the Ct method. Ct values of Cx43 amplification
were normalized to those of GAPDH amplification.
Western blot analysis and antibodies
Western blot analysis was performed for the detection of Cx43, phospho-JNK
and phospho-c-jun in rat spinal astrocytes. After treatment with the various drugs,
the cells were solubilized in radioimmunoprecipitation assay buffer with
inhibitors (100 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 µg/ml
aprotinin, 20 µg/ml leupeptin, 1mM phenylmethylsulfonyl fluoride, andphosphatase inhibitor cocktail 2 (Sigma Chemical, Co.)). The lysates were
centrifuged and the supernatant was added to Laemli’s buffer and boiled at 95°C
for 5 min. Equal amounts of protein were separated by 7.5% (for Cx43 or β-actin)or 10% (for phosphor- or total JNK or phospho-c-jun) SDS–polyacrylamide gelelectrophoresis and blotted onto nitrocellulose membranes. The membranes wereincubated with blocking solution, and subsequently incubated with a purified
polyclonal antibody against rat Cx43 (1:1000, Cell Signaling Technology, Beverly,
MA, USA)), phospho-JNK (1:1000, Cell Signaling Technology), total-JNK (1:1000,
23

Cell Signaling Technology), phospho-c-jun (1:1000, Cell Signaling Technology) orwith a monoclonal antibody against β-actin (1:10,000, Sigma Chemical, Co.)overnight at 4 °C. After being washed, the membranes were incubated with thehorseradish peroxidase-conjugated secondary antibody for 1 h at room
temperature. Thereafter, membranes were rinsed and incubated with
Luminescence reagent (Thermo Fischer Scientific, Rockford, IL, USA). Finally,
the membranes were exposed to X-ray film. For quantification of signals forproteins, the densities of specific bands were measured with Science Lab Image
Gauge (Fuji Film, Tokyo, Japan).
Scrape loading/dye transfer assay
Several assays are currently available to determine the GJIC in cultured cells
56-58.

Among these, the scrap loading/dye transfer (SLDT) assay is based onmonitoring the transfer of the fluorescent dye Lucifer yellow from one cell into
adjacent cells and has been extensively used to analyze the functional status ofgap junction in cell culture systems. The SLDT assay utilizing Lucifer yellow was
performed to examine the effect of cytokines on GJIC between spinal astrocytes
according to a previously described method 59. Briefly, cells were incubated in
HEPES-buffered salt solution for 10 min containing the following (in mM): 140
NaCl, 5.5 KCl, 1.8 CaCl2, 1 MaCl2, 10 glucose, 10 HEPES, pH 7.35. Cells werethen washed in Ca2+-free HEPES solution for 1 min, and then scrape loading wasachieved with a razor blade in the same Ca2+-free solution containing Lucifer
yellow CH (1 mg/ml; Sigma Chemical, Co). After 1 min, cells were washed several
times with the HEPES-buffered salt solution. After scrap loading for 8 min,fluorescent images were captured using an inverted fluorescent microscope
equipped for epifluorescence (Olympus, Tokyo, Japan) and images were analyzed
using image-analysis software image J (National Institutes of Health, USA). For
24

each trial, determining the fluorescence area and intensity in time consecutive
fields performed quantification of the change in GJIC induced by different
treatments.
Proteasome activity
The proteasome activity was analyzed by using a 20s proteasome assay kit
according to the manufacturer’s instructions. Briefly, seed astrocytes in a 96-wellplate at a density of 3×105 cell/well. After treatment, astrocytes were lysate by 20s
proteasome lysis buffer, then add 20s proteasome substrates SUC-LLVY-AMC to
incubate for 1 h at 37 °C. Finally, read the fluorescence intensity of AMC using an
excitation and emission wavelengths of 360nm and 480nm filter set in afluorometer (Perkin Elmer, New York, USA) epifluorescence (Olympus, Tokyo,
Japan). The AMC standard curve was generated with a dilution series of purified
Statistical analysis
Data are expressed as the mean ± SEM of at least three independent
experiments. Differences between means were statistically analyzed by one-way
analysis of variance (ANOVA) followed by post-hoc multiple comparison analysis
using the Tukey–Kramer method. The differences between vehicle and cytokines
treatment in the JNK and c-jun phosphorylation studies were analyzed using
Student’s t-test. A probability value (p) of less than 0.05 was considered to bestatistically significant.

2.4. Results
TNF-α and IFN-γ induced a down regulation of Cx43 expression in
spinal astrocytes
To elucidate the effects of TNF-α, IFN-γ and IL-1β on Cx43 expression in spinal
astrocytes, changes in Cx43 mRNA and protein expression were quantified. As
25

shown in Fig. 2-1A, Cx43 expression was significantly decreased with TNF-αtreatment at 24 h and 48 h post-treatment and with IFN-γ. Moreover,co-treatment with TNF-α and IFN-γ induced a marked decrease in Cx43 mRNA
expression at both 24 and 48 h post-treatment. By contrast, treatment with IL-1β
for either 24 or 48 h did not result any significant change in Cx43 mRNA (Fig.
2-1A).
A decrease in Cx43 protein expression was observed with treatment with either
TNF-α or IFN-γ at both 24 h and 48 h. Similar to the effect observed in Cx43mRNA expression, a robust reduction of Cx43 protein expression was obtained bya mixture TNF-α and IFN-γ at both 24 and 48 h post-treatment (Fig. 2-1B). By
contrast, treatment with IL-1β did not result in any significant change in Cx43
protein expression at either 24 h or 48 h post-treatment (Fig. 2-1C). As a mixtureof TNF-α and IFN-γ appeared to yield a greater effect on both Cx43 transcription
and translation than either cytokine alone, the effect of co-incubation with both
cytokines on Cx43 expression over time was evaluated. A significant decrease inCx43 mRNA expression in spinal astrocytes was apparent as early as 3 hpost-incubation with the cytokine mixture (Fig. 2-2A). Twelve h following cytokine
mixture treatment, Cx43 mRNA expression was reduced to approximately
one-third of that of vehicle treated astrocytes. In addition, the subsequently
reduction of Cx43 protein expression was obtained by a mixture TNF-α and IFN-γ
at 6 h post-treatment (Fig.2-2B).
TNF-α and IFN-γ induced an inhibition of GJIC in spinal astrocytes
Next, gap junction permeability in cultured spinal astrocytes was determinedusing the SLDT assay. As shown in Fig. 2-3A, Lucifer yellow was transferredthrough GJIC in vehicle-treated astrocytes, indicating functional gap junctions.
After treatment with either TNF-α or IFN-γ for 48 h, a slight reduction of gap
26

junction permeability was observed but this was not statistically significant
compared to vehicle-treated astrocytes (p>0.05). By contrast, after co-incubation
with the cytokine mixture for 48 h, gap junction permeability between spinal
astrocytes was significant reduced (61.5 ± 9.1%, n=7; p<0.01 vs. vehicle-treated;
Fig. 2-3B).
JNK plays a critical role in the inhibitory effect of TNF-α and IFN-γ on
Cx43-GJIC in spinal astrocytes
The involvement of MAPK in the down-regulation of Cx43-gap junction induced
by a mixture of TNF-α and IFN-γ was elucidated by pharmacological approaches.
Treatment with SP600125, a potent inhibitor of JNK, significantly blocked thecytokine mixture-induced decrease of both Cx43 mRNA (Fig. 2-4A) and protein
levels (Fig. 2-4B and Table 1). In contrast, treatment with either U0126 orSB202190, which are the inhibitors of ERK and p38 MAPK, respectively, had noeffect on either Cx43 mRNA (Fig. 2-4A) or protein expression (Fig. 2-4B and Table1). Moreover, the SLDT assay results demonstrated that treatment withSP600125 reversed the cytokine mixture-induced down-regulation of gap junction
function in spinal astrocytes (Fig. 2-5). In contrast, treatment with either U0126
or SB202190 had no effect on GJIC (data not shown).
These findings suggest that the JNK signaling pathway could be involved in the
TNF-α and IFN-γ induced down-regulation of both Cx43-gap junction expression
and function. Thus, phosphorylation levels of JNK were measured to determine
whether JNK was actually activated by both cytokines in cultured spinal
astrocytes. Co-treatment with both cytokines markedly induced thephosphorylation of JNK after 30 min, and this response was significantly elevatedfor at least 12 h (Fig. 2-6A). Thirty minutes pretreatment with SP600125
significantly suppressed cytokine mixture-induced JNK phosphorylation (Fig.
27

2-6B). In addition, the phosphorylation of c-jun was also analyzed to elucidate apotential effect of JNK activity. Phosphorylation of c-jun was also significantly
increased after co-treatment with both cytokines beginning 30 min after
incubation, and this response was sustained for at least 12 h (Fig. 2-6C). The
cytokine mixture-induced up-regulation of phospho-c-jun (3 h) in spinal astrocytes
was reversed by SP600125 (Fig. 2-6D).
TNF-α and IFN-γ promote the degradation of Cx43 expression in
spinal astrocytes
Furthermore, to investigate the detail mechanism of Cx43 down-regulation,next elucidate the effect of TNF-α and IFN-γ on degradation of Cx43 in spinal
astrocyte used cycloheximide plus assay. As shown in Fig. 2-7, treated withcycloheimide (1µm) alone, that a protein synthesis inhibitor caused atime-dependent decrease in Cx43 level, after 6 h the Cx43 protein levels weredecreased by 50% compared to the 0 h. However, this effect was accelerated by
TNF-α and IFN-γ. In addition, co-treatment with TNF-α and IFN-γ induced amarked acceleration in Cx43 degradation (Fig 2-7). This suggests that TNF-α andIFN-γ induced the Cx43 degradation in spinal astrocytes.
Proteasome plays a critical role in the inhibitory effect of TNF-α andIFN-γ on Cx43-GJIC in spinal astrocytes
Next, the involvement of proteasome in the down-regulation of Cx43-gap
junction induced by a mixture of TNF-α and IFN-γ was elucidated bypharmacological approaches. Treatment with MG132, a reversible proteasome
inhibitor or epoxomicin, an irreversible proteasome inhibitor, respectively,
significantly blocked the cytokine mixture-induced decrease of both Cx43 protein
level (Fig. 2-8A) and gap junction function (Fig. 2-8B) in spinal astrocytes. Thesefindings suggest that the proteasome pathway could be involved in the TNF-α and28

IFN-γ induced down-regulation of both Cx43-gap junction expression and function.
Thus, proteasome activities were measured by used 20s proteasome assay kit todetermine whether proteasome was actually activated by both cytokines incultured spinal astrocytes. Co-treatment with both cytokines significantly
increases the activity of proteasome after 4 h, and this response was significantly
elevated for at least 12 h. In addition, the cytokine mixture-induced up-regulationof proteasome activity in spinal astrocytes was reversed by SP600125 (Fig. 2-9A).
Furthermore, the inhibited role of co-treatment both JNK and proteasome
inhibitors on Cx43 down regulation induced by TNF-α and IFN-γ also
investigated. As shown in Fig. 2-9B, after co-incubation with the inhibitors
mixture did not result any significant change in Cx43 down regulation induced bycytokines-mixture compared the inhibitor single treatment in spinal astrocytes(Fig. 2-9B). These results suggest that TNF-α and IFN-γ induce the proteasome
activation via JNK activation and then decrease the Cx43-GJIC in spinal
astrocyte.
The involvement of other signaling cascades in the regulation of Cx43
expression which have been reported to be activated after incubation with TNF-α
and IFN-γ or regulated the Cx43 expression was investigated by pharmacologicalapproaches. None of the other inhibitors had a significant effect on Cx43 protein
expression (Table 2).

2.4. Discussion
The current study demonstrated that TNF-α and IFN-γ, both alone and incombination, caused a significant down-regulation of Cx43 in spinal astrocytes,
29

leading to decreased gap junction function, as demonstrated by the SLDT assay,
which in turn was mediated by JNK-proteasome activation. These results are inaccordance with a previous report that demonstrated that lipopolysaccharide(LPS) treatment of rat cortical astrocytes led to a decrease of Cx43, which was
mediated by Toll-like receptor 4 60,61. Although LPS is known to produce
proinflammatory cytokines such as TNF-α and IL-1β 60-62, there are no reports,
which directly demonstrated that TNF-α and IFN-γ trigger the reduction of Cx43
expression. In fact, this study suggests that the cytokine IL-1β has no role in the
reduction of Cx43 expression in spinal astrocytes. Thus, the current study
suggests that specific cytokines are involved in the down-regulation of Cx43
following spinal tissue pathology. Furthermore, based on the pharmacologicaleffects, the current study indicates that a specific signaling pathway is involved in
the down-regulation of Cx43 expression and function. The findings in totalsuggest a rational approach to developing treatments for spinal pathologies
involving astrocytic dysfunction.
It is known that a network of astrocytes linked by gap junctions could play animportant role in maintaining the homeostatic environment of the CNS 63.
Several cytokines have been reported to contribute to the regulation of Cx43
expression and GJIC in brain astrocytes but these may differ from those that
regulate spinal astrocyte Cx43 expression and GJIC. For example, althoughtreatment of mouse cortical astrocytes with TNF-α, IFN-γ and IL-1β alone did notaffect Cx43 and GJIC, co-treatment with TNF-α and IL-1β markedly reduced theexpression of Cx43 and GJIC 24,59,64. By contrast, in this study, although IL-1β wasineffective on Cx43 expression in spinal astrocytes, both TNF-α and IFN-γ alone
and in combination in particular exhibited prominent inhibitory effects on Cx43
expression. Furthermore, co-treatment with TNF-α and IL-1β did not exhibit30

additional decrease of Cx43 expression in spinal astrocytes (data not shown).
Certainly, it is possible that astrocytes in different region of CNS could havedistinct properties and responses to various neurochemical stimuli. A differential
effect between brain and spinal astrocytes of cytokines could form the basis oftargeted treatment for specific CNS pathologies. Currently, however, there has
not been extensive differentiation of phenotypes, between brain and spinal
astrocytes, to confirm such a possibility.
The current study is the first to demonstrate that IFN-γ could induce an
inhibitory effect on Cx43 function. IFN-γ is produced mainly by T-cells andnatural killer cells, and mediates various immune responses to tissue injury.
Recently, IFN-γ was shown to be unregulated in the spinal cords of rats with apainful peripheral neuropathy 65, in the CNS in a mouse model of amyotrophic
lateral sclerosis (ALS) 66, and traumatic brain injury 67. The expression of IFN-γ
in the CNS under various pathological states indicates that it is a ubiquitous
cytokine and functional in both brain and spinal cord. Spinal astrocytes inparticular can express the IFN-γ receptor, and are therefore sensitive to the effectof IFN-γ 68. Thus, it is concluded that IFN-γ alone has an important role inregulating Cx43 expression in spinal cord astrocytes.
Interestingly, this study found that co-treatment of TNF-α with IFN-γ further
suppressed the expression of Cx43 in spinal astrocytes as well as further
suppressing GJIC. It should be apparent that not one but several cytokines maybe
found in the CNS under pathological conditions. To better conceptualize theclinical effect of cytokines on CNS tissues, the use of combinations of cytokines
may be more useful rather than individual cytokines. The levels of both TNF-α
and IFN-γ have been shown increased in the spinal cord under pathologicalsituations, such as a model of familial ALS 66,69,70. The onset of symptoms
31

following experimental autoimmune encephalomyelitis correlates with increased
TNF-α and IFN-γ in spinal cord 71. Thus, in clinical spinal neurodegenerative
disorders, both TNF-α and IFN-γ are crucial in triggering astrocytic dysfunction.
Although it has been reported that cytokines can reduce Cx43 expression invarious cells types, it is yet unclear, which signaling mechanism underlies thecytokine-mediated reduction of Cx43 expression. In the current study, both thedysfunction of Cx43 synthesis and Cx43 degradation were involved the Cx43-gap
junction down-regulation induced by TNF-α and IFN-γ. The main focus was on
the involvement of MAPK in the TNF-α and IFN-γ induced down-regulation of
Cx43, because MAPK is known to be activated after stimulation with cytokines inspinal astrocytes 48,49. The current results indicated that JNK, but not ERK or p38,
plays a pivotal role in the TNF-α and IFN-γ induced decrease the mRNA andprotein levels of Cx43 and GJIC in spinal astrocytes. Furthermore, treatment
with both cytokines markedly induced the phosphorylation of JNK and c-jun,which precedes the down-regulation of Cx43 mRNA expression. The
phosphorylation of JNK has been observed under various pathological situations
such as neuropathic or inflammatory pain 72,73, suggesting that activation of thiskinase in astrocytes is important step in the molecular process that leads, in thiscase, to a chronic pain state. These observations, combined with the current
findings, indicate that JNK is a crucial factor in the cytokine-mediated regulationof Cx43 in spinal astrocytes. Interestingly, in current study suggested that JNK
also involved the TNF-α and IFN-γ induced Cx43 degradation via proteasome
activity. It is supported by several recent study that TNF-α or IFN-γ mediatedastrocyte activity was mediated by proteasome pathway 74,75. Furthermore, C43
degradation in human corneal fibroblasts or rat BWEM cell was also prevented byproteasome inhibitors 36,51. The proteasome system also has been observed under32

various pathological situations such as chronic pain 76. These observations,combined with the current findings, indicate that proteasome is also a crucial
factor in the cytokine-mediated regulation of Cx43 in spinal astrocytes. However,
it is also shown that Cx43 ubiquitin was significantly increased in human corneal
fibroblasts51. Thus, the ubiquitin level of Cx43 in spinal astrocyte after
treatments with both cytokines are future must be examined.
As multiple signaling molecules are involved in the regulation of brain
astrocytic Cx43, such could also be the case for spinal astrocytes. Previous studies
indicated that LPS-induced inhibitory effect on Cx43-GJIC in cortical astrocytes
was mediated through a down-regulation of membrane protein caveolin-3 61.
NF-κB and the phosphoinositide 3-kinase pathway activated by Toll-like receptor
3 were implicated in the down-regulation of astrocytic Cx43 expression in human
fetal brain 77. The molecular mechanism of the reduction of Cx43 synthesis
combined with JNK activation remains to be clarified, involving either a direct oran indirect mechanism. It is possible that JNK could directly activate atranscriptional repressor to block the expression of Cx43 mRNA, or via anepigenetic mechanism to initiate gene silencing. Indeed, accumulating evidence
has shown that epigenetic modulation is associated with Cx43 gene expression
78-80. One possible indirect mechanism is that JNK contributes to the production of

inhibitory molecules to block Cx43 transcription, thereby reducing Cx43 protein
expression. Several studies have indicated that cytokines utilize a pathway that is
highly dependent on treatment with TNF-α and IFN-γ leads to the activation ofmultiple signaling molecules, including PKA, PKC, JAK, src tyrosine kinase and
ROS 81-84. However, the current study excluded the possibility of involvement ofthese molecules by pharmacological approaches. Thus, our data indicate thatcytokines utilize a pathway that is highly dependent on JNK phosphorylation,33

thereby modulating Cx43-GJIC in spinal astrocyte.
In conclusion, the current study demonstrated that TNF-α and IFN-γ,
separately and in combination induced a down-regulation of Cx43 expression in
spinal astrocytes, which led to decreased gap junction function and that the
down-regulation is critically dependent on JNK activation and proteasome
activity. As a consequence of diminished functioning of gap junctions, there is a
failure of intercellular communication and sensing of the synaptic milieu. Under
neurodegenerative or neuroinflammatory conditions, JNK appears to have an
essential transcriptional role in dysfunctional spinal astrocytes. Indeed, JNK
appears to be involved in a number of processes in both glia and CNS neurons
following injury 73,85. In addition, the proteasome activities have an important roleon protein degradation. Further elaborating the molecular mechanism that
down-regulates the expression of Cx43 might lead to new strategies to rescue CNS
tissue in diverse neurological disorders.

34Summary
In chapter 1 demonstrated that after nerve injury spinal astrocytic
Cx43 was decreased via TNF-α and then induced GLT-1 down
regulation.

These

responses

inducedneuropathic

pain

via

glutamatergic transmission.
In chapter 2 demonstrated that treatment of spinal astrocyte with
mixture of cytokines (TNF-α, IFN-γ) led to a decrease of Cx43
expression via JNK and proteasome pathway.

Schematic illustration of the role of Cx43 on neuropathic
pain (in vivo) and the signaling pathway of Cx43 down
regulation in spinal astrocyte (in vitro).

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Table.2-1. Effect of M APK on the cytokines-m ediated down-regulation on
Cx43 protein expression in spinal astrocytes.
inhibitor

Cx43 protein expression
TNF-α + IFN-γ+Inhibitors

Inhibitors

U0125 (10 µM, P38 inhibitor)

0.294 + 0.048

0.414 + 0.091

0.940 + 0.095

SB202125 (10 µM, ERK inhibitor)

0.294 + 0.048

0.214 + 0.085

0.847 + 0.183

SP600125 (10 µM, JNK inhibitor)

0.294 + 0.048

0.667 + 0.052++ 1.083 + 0.105

The levels of Cx43 protein in spinal astrocytes incubated with or without inhibitors are
expressed as a ratio of protein levels from vehicle-treated astrocytes. Astrocytes werepretreated for 30 min with inhibitor before treatment with the cytokine mixture (TNF-α
10 ng/ml and IFN-γ 5 ng/ml). Data represent the mean + SEM for 3 independent
experiments. ++P<0.01 vs. TNF-α + IFN-γ.

Table.2-2. Effect of several kinds of inhibitors on the cytokines-m ediateddown-regulation on Cx43 protein expression in spinal astrocytes.
Inhibitor

Cx43 protein expression
TNF-α + IFN-γ+ Inhibitors

H89 (10 µM, PKA inhibitor)

0.362 + 0.012

0.357 + 0.088

Go6983 (3 µM, PKC inhibitor)

0.366 + 0.021

0.322 + 0.092

PP1 (20 µM, tyrosine kinase inhibitor)

0.357 + 0.017

0.411 + 0.137

Genistein (50 µM, tyrosine kinase

0.380 + 0.015

0.464 + 0.129

NAC (1 mM, Ros inhibitor)

0.385 + 0.111

0.459 + 0.106

AG490 (10 µM, JAK inhibitor)

0.300 + 0.027

0.338 + 0.016

inhibitor)

The levels of Cx43 protein in spinal astrocytes incubated with or without inhibitors are
expressed as a ratio of protein levels from vehicle-treated astrocytes. Astrocytes werepretreated for 30 min with inhibitor before treatment with the cytokine mixture (TNF-α
10 ng/ml and IFN-γ 5 ng/ml). Data represent the mean + SEM for at least 3 independent
experiments.

43

Figure1-1. PSN L induced change of tactile m echanical threshold and Cx43
expression in spinal dorsal horn.
After PSNL treatment, the withdrawal thresholds of the ipsilateral hind (A) and
expression of Cx43 in ipsilateral hind spinal dorsal horn were measured by von Frey test
and Western blotting, respectively for the periods indicated (3, 7, 14 or 21 day). (A) The
values are expressed as the weight of von-Frey filament. (B) In Western blots Cx43
protein levels were normalized to that of β-actin, which served as an internal control, andexpressed as ratio of the level of sham group at the corresponding time point. The data
represent the mean + SEM (bar) of at least three independent experiments. **P < 0.01 vs.
sham group.

Figure1-2. Cx43 downregulation in spinal cord induced change of tactile
m echanical threshold.
After intreathecal injected siRNA (5 pg), expressions of Cx43 in spinal dorsal horn andmechanical thresholds were measured by western blotting (A) and von-Frey test (B),respectively for the periods indicated (1, 2, 3, 5 or 7 day). After intreathecal injection ofcarbenoxolone (CBX, 5 pg), the mechanical thresholds were measured by von-Frey test
(C). The data represent the mean + SEM (bar) of at least three independent experiments.
**P < 0.01 vs. control siRNA group or saline group.
44

Figure1-3. PSN L induced change of tactile m echanical threshold and Cx43
expression in spinal dorsal horn.
The change of Cx30 expression in ipsilateral hind spinal dorsal horn at 7 and 14 days
after PSNL treated mice (A) and in spinal dorsal horn at 3 days after Cx43 siRNA treatedmice (B) were measured by Western blotting. The Cx30 protein levels were normalized tothat of β-actin, which served as an internal control, and expressed as ratio of the level ofsham group or control siRNA group. The data represent the mean + SEM (bar) of at least
three independent experiments.

Figure1-4. TNF-α involves the PSNL induced down regulation of Cx43 in spinal
dorsal horn.
The protocol of repeated-intrathecal injected with etanercept for PSNL mice (A). The
45

expression of Cx43 in the spinal dorsal horn in PSNL mice (B) or TNF-α treated mice (D) were
measured by western blotting. The change of withdrawal thresholds after etanercept
treatment mice (C) or TNF-α treated mice (E) was measured by von Frey test. The data
represent the mean + SEM (bar) of at least three independent experimens. **p<0.01 vs.
sham-saline group or saline group.

Figure1-5. Glutam ate receptors antagonists attenuate the Cx43 siRNA-inducedm echanical hypersensitivity
After treated several antagonists including MK801 (A), CNQX (B), CP96345 (C) or
cabapentin (D), the change of mechanical hypersensitivity induced by Cx43 siRNA were
measured by von Frey test.

Values are expressed as the weight of von-Frey filament. The

data represent the mean + SEM (bar) of at least three independent experiments. *p<0.05,**p<0.01 VS. sicon-saline group. +p<0.05, ++p<0.01 VS. siCx43-saline group.

46

Fig.1-6. The down-regulation of Cx43 expressions were related GLT -1 decreasingin spinal dorsal horn.
After treatment with PSNL (a) or Cx43 siRNA (b), the expression levels of GL-1 in spinal
dorsal horn were measured by Western blotting. After treatment with carbenoxolone (CBX)
the Cx43 expression (c) and GLT-1 expression (d) were also measured by Western blotting.
The data represent the mean + SEM (bar) for at least three independent experiments,**P<0.01 vs. sham group, control-siRNA group or saline group.

47

Figure1-7. The expression of GLAST in spinal dorsal horn not changed following
Cx43 down regulation.
The expression of GLAST in the ipsilateral hind spinal dorsal horn at PSNL mice (A) and inspinal dorsal horn in Cx43 siRNA treated mice (B) were measured by Western blotting. The
GLAST protein levels were normalized to that of β-actin, which served as an internal control,and expressed as ratio of the level of sham group or control siRNA group. The data represent
the mean + SEM (bar) of at least three independent experiments. **P<0.01 vs. sham group.

Fig.2-1. Effect of cytokines on the expression of Cx43 in cultured rat spinal
astrocytes.
After treatment of spinal astrocytes with either TNF-α (TNF, 10 ng/ml), IFN-γ (IFN, 5
ng/ml), IL-1β (10 ng/ml) or a mixture of TNF-α and IFN-γ (TNF+ IFN) for 24 or 48 h, theexpression levels of Cx43 mRNA (A) and protein (B) were analyzed by real-time PCR andWestern blotting, respectively. (A) The expression of Cx43 mRNA was normalized to that of
GAPDH mRNA, used as an internal control. Data are expressed as a ratio of the level ofvehicle-treated astrocytes. (B) In Western blots Cx43 protein levels were normalized to that of
β-actin, which served as an internal control, and expressed as ratio of the level ofvehicle-treated astrocytes. The data represent the mean + SEM (bar) of at least 3 independent
experiments, *P < 0.05, **P < 0.01 vs. vehicle-treated.
48

Fig.2-2. Tim e dependent effects of co-treatm ent with TNF-α and IFN-γ on Cx43
m RNA and protein levels.
Spinal astrocytes were treated with a mixture of TNF-α (TNF, 10 ng/ml) and IFN-γ (IFN, 5
ng/ml) for the periods indicated (0.5, 1, 3, 6, 9 or 12 h). The expressions level of Cx43 mRNA(A) and protein (B) were measured by real-time PCR and Western blotting, respectively. Data
represent the mean + SEM (bar) at least 3 independent experiments, **P < 0.01 vs.
vehicle-treated.

Fig.2-3. Effect of co-treatm ent with TNF-α and IFN-γ on the function of GJIC in
cultured spinal astrocytes.
Spinal astrocytes were treated with either TNF-α (TNF, 10 ng/ml), IFN-γ (IFN, 5 ng/ml) or
both for 48 h. After incubation, cells were cut with a razor blade in the presence of Lucifer
yellow. (A) Spreading of the dye was taken as an indicator for intercellular junction coupling.
Scale bar, 100 µm. (B) The graph represents the quantitation of the spreading fluorescentarea × intensity. The values are expressed as a ratio of vehicle-treated astrocytes. The data
represent the mean + SEM (bar) for at least 3 independent experiments, **P<0.01 vs.
vehicle-treated.
49

Fig.2-4. Effect of M AP kinase inhibitors on cytokine-m ediated down-regulationof Cx43 in cultured spinal astrocytes.
After treatment with U0126 (U, 10 µM), SB202190 (SB, 10 µM) or SP600125 (SP, 10 µM) for
30 min, spinal astrocytes were stimulated with a mixture of cytokines (TNF-α 10 ng/ml, IFN-γ
5 ng/ml) for 48 h. The expression levels of Cx43 mRNA and protein were measured byreal-time PCR (A) and Western blotting (B), respectively. The data represent the mean +SEM (bar) for at least 3 independent experiments, **P<0.01 vs. vehicle-treated. +P<0.05 vs.
TNF-α + IFN-γ. ++P<0.01 vs. TNF-α + IFN-γ.

Fig.2-5. Effect of JNK inhibitors on cytokine-m ediated down-regulation of GJIC
in cultured spinal astrocytes
After incubation with SP600125 (SP, 10 µM) for 30 min, cells were stimulated with amixture of TNF-α (TNF, 10 ng/ml) and IFN-γ (IFN, 5 ng/ml) for 48 h. Then, GJIC between
spinal astrocytes was measured by the SLDT assay. Upper panels indicate representative50

results. Scale bar, 100 µm. The graph in the lower part of figure 6 is quantitation of the
spreading fluorescent area × intensity. The values are expressed as a ratio of vehicle-treated.
The data represent the mean + SEM (bar) for at least 3 independent experiments, **P<0.01
vs. vehicle-treated. ++P<0.01 vs. TNF-α + IFN-γ.

Fig.2-6. Effect of co-treatm ent with TNF-α and IFN-γ on JNK and c-jun
phosphorylation levels in cultured spinal astrocytes.
The time-courses (hours) of JNK phosphorylation (A) and c-jun phosphorylation (C) after
co-incubation with TNF-α (TNF, 10 ng/ml) and IFN-γ (IFN, 5 ng/ml) are shown. Upper panel
are representative blots. The graph in the lower part of the figure represents quantitative
data for each blot. Each level of JNK phosphorylation was normalized for total JNK and c-jun
phosphorylation was normalized for β-actin and expressed as a ratio of vehicle-treated levels.
The effect of SP600125 on the phosphorylation levels of JNK (B) after stimulation with both
cytokines for 30 min and phosphorylation levels of c-jun (D) after stimulation with both
cytokines for 3 h are shown. Data represent the mean + SEM (bar) for at least 3 independent
experiments, *P < 0.05, **P < 0.01 vs. vehicle-treated, +P<0.05 vs. TNF-α + IFN-γ, ++P<0.01 vs.
TNF-α + IFN-γ.
51

Figure2-7. Effect of TNF-α and IFN-γ on the Cx43 degradation in spinal
astrocytes.
Spinal astrocytes treated with cycloheximide (1µM) for the periods indicated (0, 2, 4 or 6 h)after 3 h treated TNF-α (TNF, 10 ng/ml), IFN-γ (IFN, 5 ng/ml) or TNF-α+IFN-γ (TNF+IFN)(A). The expression levels of Cx43 protein were measured by Western blotting (B). Data
represent the mean + SEM (bar) at least 3 independent experiments, *P < 0.05 vs.
vehicle-treated at respectively h.

Fig.2-8. Effect of proteasom e inhibitors on cytokine-m ediated down-regulation of
Cx43-gap junction in cultured spinal astrocytes.
After treatment with either MG132 (MG, 5 µM) or epoxomicin (epoxo, 25 nM) for 1 h, spinal
astrocytes were stimulated with a mixture of cytokines (TNF-α, TNF 10 ng/ml, IFN-γ, IFN 5
ng/ml) for 24 h. The expression levels of Cx43 protein and gap junction function were
measured by Western blotting (A) and SLDT assay (B), respectively. The data represent themean + SEM (bar) for at least 3 independent experiments, **P<0.01 vs. vehicle-treated.
+P<0.05 vs. TNF-α + IFN-γ. ++P<0.01 vs. TNF-α + IFN-γ

52

Fig.2-9. The JNK activity plays an im portant role in the cytokines-induced
proteasom e activity.
After treatment with SP600125 for 30 min, astrocytes were treated a mixture of cytokines(TNF-α, TNF 10 ng/ml, IFN-γ, IFN 5 ng/ml) for 4, 8 or 12 h. the proteasome activity wasmeasured by using 20s proteasome assay kit (A). After treatment with either SP600125 (SP,
10 µM), MG132 (MG, 5 µM), epoxomicin (epoxo, 25 nM), a mixture of SP600125+MG132 or a
mixture of SP600125+epoxomicin for 1 h, cells were stimulated with a mixture of cytokinesfor 24 h. Data represent the mean + SEM (bar) for at least 3 independent experiments, *P <0.05, **P < 0.01 vs. vehicle-treated, +P<0.05 vs. TNF-α + IFN-γ, ++P<0.01 vs. TNF-α + IFN-γ.

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