Available via license: CC BY 2.0
Content may be subject to copyright.
R E S E A R C H Open Access
Connexin43 mediates NF-κB signalling activation
induced by high glucose in GMCs: involvement
of c-Src
Xi Xie
1,2
, Tian Lan
3
, Xiuting Chang
1
, Kaipeng Huang
1
, Juan Huang
1
, Shaogui Wang
1
, Cheng Chen
1
, Xiaoyan Shen
1
,
Peiqing Liu
1
and Heqing Huang
1*
Abstract
Background: Nuclear factor kappa-B (NF-κB) signalling plays an important role in diabetic nephropathy. Altered
expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. The aim of the current study was
to investigate the role of Cx43 in the activation of NF-κB induced by high glucose in glomerular mesangial cells
(GMCs) and to determine whether c-Src is involved in this process.
Results: We found that downregulation of Cx43 expression induced by high glucose activated NF-κB in GMCs.
Orverexpression of Cx43 attenuated NF-κB p65 nuclear translocation induced by high glucose. High glucose
inhibited the interaction between Cx43 and c-Src, and enhanced the interaction between c-Src and IκB-α. PP2,
a c-Src inhibitor, also inhibited the tyrosine phosphorylation of IκB-αand NF-κB p65 nuclear translocation induced
by high glucose. Furthermore, overexpression of Cx43 or inhibition of c-Src attenuated the upregulation of
intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN)
expression induced by high glucose.
Conclusions: In conclusion, downregulation of Cx43 in GMCs induced by high glucose activates c-Src, which in
turn promotes interaction between c-Src and IκB-αand contributes to NF-κB activation in GMCs, leading to renal
inflammation.
Keywords: Connexin43, NF-κB signalling, c-Src, Diabetic nephropathy, Inflammation, Fibronectin
Background
Diabetic nephropathy (DN) is one of the most serious
microvascular complications of diabetes and the leading
cause of end-stage renal failure. DN is characterized by ex-
cessive deposition of extracellular matrix (ECM) proteins,
such as fibronectin (FN) and collagen, in the glomerulus
and renal tubulointerstitium [1,2]. Hyperglycemia is the
primary pathogenetic factor for diabetic renal diseases. In
recent years, inflammation has emerged as a key patho-
physiological mechanism of DN. Chronic low-grade in-
flammation and activation of the innate immune system
play significant roles in the pathogenesis of DN [3,4]. In
diabetes, activated nuclear factor-κB(NF-κB) translocates
into the nucleus and triggers the expression of its target
genes, including intercellular adhesion molecule-1 (ICAM-
1) and transforming growth factor-beta 1 (TGF-β1). These
genes cause persistent and enhanced inflammation, leading
to excessive FN production and ECM accumulation. Con-
sequently, the pathogenesis of glomerular sclerosis and
tubulointerstitial fibrosis are accelerated [5-7]. However,
the mechanisms by which high glucose activates NF-κBin
DN remain to be explored.
Gap junctional intercellular communication (GJIC) re-
lies on the presence of intercellular protein channels that
span the lipid bilayers of contiguous cells, allowing them
to directly exchange ions and small molecules [8]. In ver-
tebrates, gap junctions are comprised of a multi-gene fam-
ily called connexins, among which connexin43 (Cx43) is
expressed the most extensively [9]. Glomerular mesangial
cells (GMCs) are highly coupled by Cx43-containing gap
* Correspondence: huangheq@mail.sysu.edu.cn
1
Laboratory of Pharmacology & Toxicology, School of Pharmaceutical
Sciences, Sun Yat-sen University, Guangzhou 510006, China
Full list of author information is available at the end of the article
© 2013 Xie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Xie et al. Cell Communication and Signaling 2013, 11:38
http://www.biosignaling.com/content/11/1/38
Figure 1 (See legend on next page.)
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 2 of 17
http://www.biosignaling.com/content/11/1/38
junctions. Expression level of Cx43 has been reported to
parallel the function of GJIC [10,11]. Several studies have
demonstrated that Cx43 is involved in the pathogenesis of
DN. For example, protein level of Cx43 has been reported
to decrease in the kidneys of diabetic patients and animals.
Altered gap junctional communication, including abnor-
mality in Cx43, plays a role in altered renal auto-regulation
in diabetes [12,13]. Decreased Cx43 is also found in high
glucose-treated GMCs. Downregulation of Cx43 induced
by high glucose results in senescence and hypertrophy of
GMCs [11,14].
The intracellular carboxy tail of Cx43 (Cx43CT) inter-
acts with numerous signalling and scaffolding proteins
and thereby regulates cell functions such as cell adhe-
sion, migration, and proliferation [8,15]. Cx43CT inter-
acts with c-Src, a non-receptor tyrosine kinase that can
regulate cell proliferation. Activated c-Src phosphory-
lates Cx43 on the critical tyrosine residues, Tyr247 and
Tyr265, and reduces intercellular communication and
Cx43 internalisation [16,17]. High glucose-induced pro-
tein kinase C and c-Src-dependent big mitogen-activated
protein kinase 1 activation are reportedly involved in
the pathogenesis of DN [18]. A recent study has shown
that activation of c-Src mediates platelet-derived growth
factor-induced smad1 phosphorylation and contributes
to the progression of glomerulosclerosis in glomerulo-
nephritis [19].
As mentioned above, decreased Cx43 and activated c-
Src, which interacts with Cx43CT, are associated with
the pathogenesis of DN. Here, we investigated the role
of Cx43 in the activation of NF-κB induced by high glu-
cose in GMCs to determine whether c-Src is involved in
this process. In addition, we elucidated the molecular
mechanism linking these cellular events.
Results
Cx43 expression is downregulated and c-Src activity is
enhanced in the kidneys of diabetic animals and GMCs
exposed to high glucose
We examined expression of Cx43 in diabetic kidneys
of diabetic (db/db) mice and STZ-induced diabetic rats
by immunoblotting. Compared with normal animals,
phosphorylated form of Cx43 and total Cx43 protein levels
were reduced in the kidneys of both diabetic animals
(P<0.05; Figures 1A and B). Immunohistological staining
also showed lower positive expression of Cx43 in the kid-
neys of STZ-induced diabetic rats compared with normal
rats (Figure 1C). Double immunolabeling of frozen kidney
sections showed that Cx43 is expressed in both mesangial
and endothelial cells. Furthermore, downregulation of Cx43
was observed in both cell types in the kidneys of STZ-
induced diabetic rats (Figure 1D). High glucose (30 mmol/L)
treatment for 30 min decreased Cx43 expression in GMCs,
whereas mannitol (30 mmol/L) treatment for the same dur-
ation exhibited no such effect (Figure 1F). Immunofluores-
cence results confirmed the decrease in Cx43 expression in
GMCs cultured in 30 mM glucose (Figure 1E). c-Src Y416
phosphorylation was found to be upregulated in the kidneys
of db/db mice and STZ-induced diabetic rats, and the total
amount of c-Src remained constant throughout the experi-
ment (P<0.05; Figures 2A and B). In addition, high glucose
induced significant increase in c-Src Y416 phosphorylation
in GMCs but not in the total amount of c-Src (Figure 2C).
Cx43 and c-Src are responsible for NF-κB activation
induced by high glucose in GMCs
Cx43 is known to be regulated by NF-κB [20,21]. There-
fore, we sought to determine whether NF-κB is regulated
by Cx43 in GMCs exposed to high glucose. We transfected
GMCs with plasmids expressing Cx43-siRNA and GFP-
Cx43, and analyzed Cx43 expression by immunoblotting.
Our results showed that Cx43 expression was decreased
by about 70% after Cx43-siRNA transfection, but increased
by about 80% after GFP-Cx43 transfection. The empty
vector had no effect on Cx43 expression. Immunofluores-
cence images of GFP-Cx43-transfected cells are shown in
Figure 3B (a). Interestingly, nuclear translocation of NF-κB
p65 by high glucose and Cx43-silencing was maintained
in normal glucose. Furthermore, overexpression of Cx43
using GFP-Cx43 plasmid decreased NF-κB p65 activity
in the nuclei of GMCs cultured in high glucose (P<0.05;
Figure 3A). Immunofluorescence images also showed that
high glucose and Cx43-siRNA transfection enhanced NF-
κB p65 nuclear translocation while GFP-Cx43-transfection
(See figure on previous page.)
Figure 1 Cx43 expression is decreased in diabetic kidneys and high glucose-induced GMCs. (A, B) Phosphorylation of Cx43 and total
Cx43 expression in kidneys of db/db mice and STZ-induced diabetic rats were detected by immunoblotting. (C) Cx43 expression was measured
by immunohistochemistry in kidneys of STZ-induced rats. Staining without the Cx43 antibody was used as a negative control. (D) Images of
frozen kidney sections from STZ-induced diabetic rats stained doubly with anti-Cx43 antibody and anti-Thy-1.1 antibody (a) or anti-RECA-1
antibody (b). Red fluorescence indicates Cx43. Green fluorescence indicates thy-1.1 (a) or RECA-1 (b). Blue fluorescence indicates nuclei. Scale bar
represents 10 μm (magnification 400×). (E) GMCs were cultured in DMEM containing normal glucose (NG; 5.5 mmol/L) and serum starved for
16 h before exposure to high glucose (HG; 30 mmol/L). Cx43 expression was measured by immunofluorescence after 30 min of HG stimulation
(upper panel). Phase contrast views are also shown (second panel). Green fluorescence indicates Cx43. Scale bar represents 100 μm
(magnification 100×). Scale bar represents 20 μm in magnification views (lower panel, magnification 400×). (F) Cx43 was measured by
immunoblotting after treatment for 30 min with high glucose (30 mmol/L). Mannitol (30 mmol/L) was used as an osmotic control. Experiments
were performed at least three times with similar results.
*
P<0.05 vs. control group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 3 of 17
http://www.biosignaling.com/content/11/1/38
inhibited high glucose-induced NF-κB p65 nuclear trans-
location (Figure 3B (b)).
c-SrcisreportedlyinvolvedinNF-κB activation [22,23].
In a previous study, we showed that high glucose induces
nucleus translocation NF-κB p65 [24]. In the current
study, we found that preincubation with PP2 (10 μM), an
inhibitor of c-Src, prevented the increase in NF-κBp65in
the nuclei induced by high glucose (P<0.05; Figure 4A).
Furthermore, PP2 also prevented nuclear translocation
of NF-κB induced by Cx43 siRNA, suggesting the import-
ant role of c-Src in NF-κB activation induced by Cx43
(P<0.05; Figure 4B). PP2 also inhibited the upregulation
of ICAM-1, TGF-β1, and FN expression induced by high
glucose in GMCs (P<0.05; Figure 4C). An inactive analogue
PP3 was used as a control and showed no effect.
High glucose induces dissociation between Cx43 and
c-Src and enhances interaction between c-Src and IκB-α
in GMCs
Given the observations above, we further investigated
the molecular mechanisms by which Cx43 mediates NF-
κB signalling in GMCs exposed to high glucose. The re-
lationships among Cx43, c-Src and IκB-αwere investi-
gated by co-immunoprecipitation and immunoblotting.
Co-immunoprecipitation results revealed that high glu-
cose decreased Cx43 and induced dissociation between
Cx43 and c-Src (P<0.05; Figure 5A). Y416 c-Src expres-
sion was also increased without changes in the total
amount of c-Src by high glucose (P<0.05; Figure 5B).
Furthermore, direct interaction between c-Src and IκB-α
and tyrosine phosphorylation of IκB-αwere observed
(P<0.05; Figure 5C). All of these changes were observed
at 15 min of high glucose treatment and persisted for at
least 120 min. Serine phosphorylation of IκB-αand deg-
radation of IκB-αwere also observed by immunoblotting
at 90 min of high glucose treatment, later than the
emergence of NF-κB p65 nuclear translocation (P<0.05;
Figure 5D).
Immunofluorescence images show the locations of Cx43,
c-Src, and IκB-αin GMCs
We next performed immuofluorescence staining of
Cx43, c-Src, and IκB-αin GMCs to confirm our co-
immunoprecipitation results. Zonula occludens-1 (ZO-1),
originally identified as a component of tight junctions, is a
member of the membrane-associated guanylate kinase
family of proteins that interacts with Cx43 at the plasma
membrane [25]. Cx43 and ZO-1 were found to co-localize
at the membrane of GMCs cultured in normal glucose.
However, a significant decrease in the membrane Cx43 of
GMCs was observed after 30 min of high glucose treat-
ment (Figure 6A (a)). c-Src was also found to be located
on the membrane of GMCs cultured in normal glucose.
High glucose induced its translocation to the cytoplasm
Figure 2 c-Src activity is upregulated in diabetic kidneys and
high glucose-induced GMCs. (A, B) c-Src activity in kidneys of db/
db mice and STZ-induced diabetic rats was detected by
immunoblotting. (C) c-Src activity in GMCs was measured by
immunoblotting for phosphorylation of Tyr416 on c-Src after
treatment for 30 min with high glucose (30 mmol/L) and reprobed
with an anti-c-Src antibody as a loading control. Mannitol (30 mmol/
L) was used as an osmotic control. Experiments were performed at
least three times with similar results.
*
P<0.05 vs. control group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 4 of 17
http://www.biosignaling.com/content/11/1/38
Figure 3 (See legend on next page.)
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 5 of 17
http://www.biosignaling.com/content/11/1/38
(Figure 6A (b)). Furthermore, an abundance of Cx43 and
the majority of c-Src were found to localize on the mem-
brane of GMCs maintained in normal glucose (Figure 6B
(a)). No co-localization was observed between c-Src
and IκB-α(Figure 6B (b)). However, Cx43 expression
on the membrane decreased after high glucose treat-
ment (Figure 6B (a).) Meanwhile, c-Src was translocated
to the cytoplasm of GMCs, where it interacted with IκB-α
(Figure 6B (b)).
Cx43 and c-Src regulate tyrosine phosphorylation of IκB-α
induced by high glucose in GMCs
To determine whether regulation of NF-κB by Cx43 and
c-Src involves tyrosine phosphorylation of IκB-α, plas-
mids of Cx43-siRNA and GFP-Cx43, and PP2, a c-Src
inhibitor, were used. High glucose alone and transfection
of Cx43-siRNA induced tyrosine phosphorylation of IκB-α
and interaction between c-Src and IκB-αin GMCs cul-
tured in normal glucose. However, pretreatment with PP2
(10 μM) significantly inhibited tyrosine phosphorylation of
IκB-αinduced by high glucose (P<0.05; Figure 7A). Res-
toration of Cx43 by transfection of GFP-Cx43 decreased
tyrosine phosphorylation of IκB-αand interaction be-
tween c-Src and IκB-αinduced by high glucose (P<0.05;
Figure 7B).
Cx43CT plays an important role in the regulation of
NF-κB by Cx43 independent of GJIC
Flag-Cx43CT, which consists of the intracellular carboxy
tail of Cx43 tagged with FLAG, was used to determine
whether regulation of NF-κB by Cx43 is independent of
GJIC. Results of scrape-loading experiments showed that
GJIC inhibited by high glucose was restored by trans-
fection of GFP-Cx43. Flag-Cx43CT did not show any
effect (Figure 8A). Like GFP-Cx43, transfection with Flag-
Cx43CT also significantly inhibited high glucose-induced
NF-κB p65 nuclear translocation (P<0.05; Figures 8B). Add-
itionally, transfection with Flag-Cx43CT exhibited an in-
hibitory effect on c-Src activation induced by high glucose
in GMCs. Our observation of co-immunoprecipitation be-
tween c-Src and FLAG suggests a direct interaction be-
tween Flag-Cx43CT and c-Src (P<0.05; Figures 8C).
Cx43 inhibits upregulation of ICAM-1, TGF-β1, and FN
expression induced by high glucose in GMCs
ICAM-1 and TGF-β1 are well-known important inflam-
matory factors in the pathogenesis of DN [26-30]. FN is
an important ECM component in the kidney [1,2]. Treat-
ment with high glucose for 24 h markedly increased
ICAM-1, TGF-β1, and FN protein levels compared with
the control group (P<0.05; Figure 9A). However, GFP-
Cx43 or Flag-Cx43CT-transfection in high glucose-treated
GMCs significantly inhibited upregulation of these pro-
teins. Transfection with the vector alone had no effect
on the production of ICAM-1, TGF-β1, and FN proteins
(P<0.05; Figure 9A). Cx43-siRNA had similar effects as
high glucose for FN, ICAM-1 and TGF-β1. Restoration of
Cx43 by transfection of GFP-Cx43 attenuated FN, ICAM-
1andTGF-β1accumulation(P<0.05; Figure 9B). Im-
munofluorescence staining also revealed that FN was
upregulated by high glucose or Cx43-siRNA, and restor-
ation of Cx43 by GPF-Cx43-transfection attenuated FN
upregulation induced by high glucose (Figure 9C).
Discussion
Downregulation of Cx43 protein expression has been
observed in the kidneys of diabetic animals and high
glucose-treated GMCs [11-14]. Consistent with previous
studies, we observed that the protein level of Cx43 was
reduced in the kidneys of db/db mice and STZ-induced
diabetic rats. Furthermore, significantly reduced Cx43
protein level was observed after 30 min of high glucose
exposure in GMCs. Previous studies have reported that
the half-life of Cx43 is short- as litter as 1–2 hours
[31-33]. We explored the half-life of Cx43 in GMCs cul-
tured in normal glucose or high glucose using cyclohexi-
mide. A significant decrease in Cx43 was observed after
30 min of normal glucose (5.5 mM) exposure. However,
high glucose (30 mM) induced a faster decrease in Cx43
after 15 min stimulation, suggesting Cx43 is actively de-
graded (Additional file 1: Figure S1). In our previous
study, we found that NF-κB signalling is activated in the
kidneys of diabetic rats and high glucose-treated GMCs
[24]. While several studies have investigated the relation-
ship between Cx43 and NF-κB signalling, most of them
have focused only on the regulation of Cx43 by NF-κB.
(See figure on previous page.)
Figure 3 Cx43 regulates high glucose-induced NF-κB nuclear translocation. (A) GMCs were transfected with Cx43-siRNA or GFP-Cx43 in
normal glucose (NG; 5.5 mmol/L). After 48 h of transfection, GMCs were exposed to high glucose (HG; 30 mmol/L) for 30 min. Proteins were then
extracted for analysis of Cx43 expression and NF-κB p65 nuclear translocation by immunoblotting. (B) Immunofluorescence images of GFP were
captured under a laser scanning confocal microscope to assess the transfection efficiency of GFP-Cx43. Green fluorescence indicates GFP. Scale
bar represents 100 μm (magnification 100×, a). Immunofluorescence images stained doubly with anti-Cx43 antibody and anti-NF-κB p65 antibody
were captured under a laser scanning confocal microscope (magnification 400×, b). High glucose and Cx43-siRNA-transfection enhance NF-κB
p65 nuclear translocation and GFP-Cx43-transfection inhibits high glucose-induced NF-κB p65 nuclear translocation. Red fluorescence indicates
localization of NF-κB p65. Green fluorescence indicates localization of Cx43. Blue fluorescence indicates nuclei. Scale bar represents 20 μm.
Experiments were performed at least three times with similar results.
*
P<0.05 vs. normal glucose-treated group,
#
P<0.05 vs. 30 mmol/L
glucose-treated group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 6 of 17
http://www.biosignaling.com/content/11/1/38
Figure 4 c-Src regulates high glucose or Cx43-induced NF-κB p65 nuclear translocation. (A) GMCs were preincubated and maintained in
10 μM PP2 (c-Src inhibitor) or 10 μM PP3 (inactive analogue) for 30 min and until the end of the experiment. Cells were then incubated in
normal glucose (NG; 5.5 mmol/L) or high glucose (HG; 30 mmol/L) for 30 min. Proteins were extracted for analysis of NF-κB p65 nuclear
translocation by immunoblotting. (B) GMCs were transfected with Cx43-siRNA in normal glucose (NG; 5.5 mmol/L). After 48 h of transfection,
GMCs were co-incubated with PP2 or PP3 (10 μM) for 30 min. Proteins were then extracted for analysis of Cx43 expression and NF-κB p65 nuclear
translocation by immunoblotting. (C) GMCs were co-incubated with 10 μM PP2 (c-Src inhibitor) or 10 μM PP3, maintained in high glucose for
24 h and then the proteins were extracted for analysis of FN, ICAM-1, and TGF-β1 by immunoblotting. Experiments were performed at least three
times with similar results.
*
P<0.05 vs. normal glucose-treated group,
#
P<0.05 vs. 30 mmol/L glucose-treated group.
**
P<0.05 vs. Cx43-siRNA
transfected group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 7 of 17
http://www.biosignaling.com/content/11/1/38
Figure 5 High glucose induces dissociation of Cx43 and c-Src, and promotes interaction between c-Src and IκB-α.GMCs were
incubated in normal glucose (NG; 5.5 mmol/L) or high glucose (HG; 30 mmol/L) for the indicated times. (A) Cx43 was immunoprecipitated with an
anti-Cx43 antibody and c-Src was analyzed by immunoblotting. white triangles=Cx43; black squares=c-Src. (B) Phosphorylation of tyr416 on c-Src
(Y416-c-Src) and total c-Src were analyzed by immunoblotting. α-Tubulin was measured by immunoblotting as a loading control. (C)IκB-αwas
immunoprecipitated with an anti-IκB-αantibody and Tyr-phosphorylation of IκB-αand c-Src were analyzed by immunoblotting. black circle= p-Tyr;
black squares=c-Src; black triangles=IκB-α.(D) Ser-phosphorylation of IκB-αand total IκB-αwere analyzed by immunoblotting. black circle=p-Ser-IκB-α;
black triangles=IκB-α.α-Tubulin was measured by immunoblotting as a loading control. Experiments were performed at least three times with similar
results.
*
P<0.05 vs. normal glucose-treated group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 8 of 17
http://www.biosignaling.com/content/11/1/38
Figure 6 (See legend on next page.)
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 9 of 17
http://www.biosignaling.com/content/11/1/38
For instance, AngII has been found to induce binding of
NF-κB to the Cx43 gene promoter, increasing Cx43 ex-
pression in aortic smooth muscle cells while the TLR3 lig-
and polyI:C has been observed to induce downregulation
of Cx43 by a mechanism involving NF-κB [20,21].
In the present study, we found that downregulation of
Cx43 induced by high glucose or transfection with the
Cx43-siRNA plasmid enhanced nuclear translocation of
NF-κB p65. However, restoration of Cx43 expression by
transfection with GFP-Cx43 attenuated high glucose-
induced NF-κB p65 nuclear translocation in GMCs,
which suggests that decreased Cx43 expression mediates
NF-κB activation in GMCs. Thus, our findings show that
Cx43 participates in the activation of NF-κB in high
glucose-treated GMCs and enhances the relationship be-
tween NF-κB and Cx43. The molecular mechanism of
this cellular event, however, remains unclear.
We also observed upregulation of c-Src activity in the
kidneys of db/db mice and STZ-induced diabetic rats. Pre-
vious studies have shown that high glucose can activate c-
Src [34,35]. Consistent with such findings, our results
show that c-Src is activated in high glucose-treated GMCs.
c-Src has been proposed to be responsible for the patho-
genesis of DN. We used PP2, a c-Src inhibitor, to explore
whether c-Src is involved in the high glucose-induced acti-
vation of NF-κB signalling in GMCs. We found that PP2
inhibited NF-κB p65 nuclear translocation induced by
high glucose or Cx43 silencing, suggesting the important
role of c-Src in Cx43-induced NF-κB activation.
As mentioned above, both Cx43 and c-Src are involved
in the activation of NF-κB in high glucose-treated GMCs.
Therefore, we further explored the molecular mechanisms
involved in these events. Previous studies have indicated
that phosphorylation of Cx43 by c-Src reduces gap junc-
tional communication depending on the interaction be-
tween Cx43CT and c-Src [17,36]. Interestingly, recent
studies have suggested that the interaction between Cx43
and c-Src reciprocally modulates their activities. The level
of Cx43 expression is important in regulating c-Src activ-
ity. Upregulation of Cx43 in glioma cells reduces c-Src ac-
tivity while silencing of Cx43 activates c-Src in astrocytes
[37,38]. In our study, reduction of Cx43 protein level in-
duced by high glucose was accompanied by decrease in
the amount of c-Src interacting with Cx43, thereby in-
creasing the activity of c-Src in the cytoplasm. This finding
indicates that downregulation of Cx43 by high glucose ac-
tivates c-Src.
The molecular mechanism by which c-Src regulates
NF-κB has been suggested to be dependent on the in-
teraction between c-Src and IκB kinase β(IKKβ)orIκB-
α. IKKβis phosphorylated by c-Src, which is involved
in TNF-α-induced ICAM-1 expression [22]. Tyrosine
phosphorylation of IκB-αactivates NF-κB through a
redox-regulated and c-Src-dependent mechanism follow-
ing hypoxia/reoxygenation [23]. In the current study,
IκB-αwas found to interact with c-Src after exposure of
GMCs to high glucose for 15 min, and to be accompan-
ied by tyrosine phosphorylation of IκB-α, persisting for
at least 120 min. We have previously shown that NF-κB
p65 is translocated into the nucleus after exposure of
GMCs to high glucose levels for 30 min [24]. Interest-
ingly, IKK-mediated serine phosphorylation of IκB-α,a
classic pathway of NF-κB activation [39], was detected
after exposure of GMCs to high glucose levels for 90
min, and this was accompanied by degradation of IκB-α,
which occurs after NF-κB p65 nuclear translocation.
Thus, tyrosine phosphorylation of IκB-αcould possibly
play an important role in the initial step of high glucose-
induced NF-κB p65 activation. As described in a previ-
ous study, tyrosine phosphorylation activates NF-κB
without degradation of IκB-α[40]. We did not observe
degradation of IκB-αwhen NF-κB p65 was translocated
into the nucleus at early stages of exposure of GMCs to
high glucose.
Immunofluorescence images showed that Cx43 and
c-Src were co-localized around the cell membrane in
GMCs maintained in normal glucose. There was no
interaction between c-Src and IκB-αin GMCs cultured
in normal glucose. However, co-localization of c-Src and
IκB-αwas observed in the cytoplasm after exposure of
GMCs to high glucose for 30 min. Based on these data,
we propose that decrease in Cx43 expression enhances
(See figure on previous page.)
Figure 6 Immunofluorescence images of co-localization of Cx43, c-Src and IκB-αin GMCs. GMCs were incubated in normal glucose (NG;
5.5 mmol/L) or high glucose (HG; 30 mmol/L) for 30 min. (A(a)) Confocal microscopy was used to evaluate the localization of Cx43 under normal
glucose or high glucose conditions. Green fluorescence indicates localization of Cx43. Red fluorescence indicates ZO-1, which served as a
cytomembrane marker. Blue fluorescence indicates nuclei. (A(b)) Confocal microscopy was used to evaluate the localization of c-Src under
normal glucose or high glucose conditions. Red fluorescence indicates c-Src. Green fluorescence indicates localization of ZO-1. Blue fluorescence
indicates nuclei. (B(a)) Confocal microscopy was used to evaluate the localization of Cx43 and c-Src under normal glucose or high glucose
conditions. A significant decrease in Cx43 was observed after high glucose treatment. c-Src dissociated from Cx43 and translocated into the
cytoplasm after high glucose treatment. Green fluorescence indicates localization of Cx43. Red fluorescence indicates c-Src localization. Blue
fluorescence indicates nuclei. (B(b)) Confocal microscopy was used to evaluate the localization of c-Src and IκB-α. c-Src was translocated into the
cytoplasm from the cytomembrane after high glucose treatment and then co-localized with IκB-αin the cytoplasm of GMCs. Green fluorescence
indicates localization of IκB-α. Red fluorescence indicates c-Src localization. Blue fluorescence indicates nuclei. Scale bar represents 20 μm
(magnification 630×).
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 10 of 17
http://www.biosignaling.com/content/11/1/38
the activity of c-Src by acting as a substrate of the kin-
ase, which promotes interaction between c-Src and IκB-
αand leads to NF-κB activation. The results of our study
confirm that PP2, an inhibitor of c-Src, can inhibit the
tyrosine phosphorylation of IκB-αand translocation of
NF-κB p65 into the nucleus, which suggests that c-Src
regulates NF-κB by inducing tyrosine phosphorylation of
IκB-α.
A recent study has reported that silencing Cx43 acti-
vates c-Src, which in turn upregulates HIF-1αleading to
the upregualtion of the machinery required to take up
glucose in astrocytes [38]. Thus, c-Src is an important
factor in the regulation of nuclear transcription factors
by Cx43. In this study, we found that high glucose and
silencing of Cx43 induced c-Src activation and promoted
interaction between c-Src and IκB-αin GMCs cultured
in normal glucose. Restoration of Cx43 greatly attenu-
ated these changes in GMCs cultured in high glucose,
confirming that the interaction between c-Src and IκB-α
is regulated by Cx43. We also explored the relationship
of HIF-1αand Cx43 in GMCs. HIF-1αprotein level was
upregulated by high glucose or reduced Cx43 level in
GMCs. Inhibition of c-Src or NF-κB abrogated the in-
crease in HIF-1αprotein level induced by high glucose.
The increase in HIF-1αprotein level was associated with
significant accumulation of FN, ICAM-1 and TFG-β1in
GMCs exposed to high glucose, suggesting a potential
role of HIF-1αin the pathogenesis of DN. However, fur-
ther research is needed to define the role of HIF-1αin
DN (Additional file 2: Figure S2).
The regulation of NF-κB by reduced Cx43 protein
level could be caused by absence of Cx43 function (gap
junctional communication) or absence of Cx43 interac-
tions with other proteins, such as c-Src. Restoration of
Cx43CT, a non-channel forming region, increases the
expression of the intracellular carboxy tail of Cx43 with-
out affecting GJIC [37]. Consistent with previous ob-
servations, our results showed that restoration of Cx43
Figure 7 Cx43 and c-Src are responsible for high glucose-
induced Tyr-phosphorylation of IκB-α.(A) GMCs were
preincubated and maintained in 10 μM PP2 (c-Src inhibitor) or 10
μM PP3 (inactive analogue of PP2) for 30 min and until the end of
the experiment. Cells were then incubated in normal glucose (NG;
5.5 mmol/L) or high glucose (HG; 30 mmol/L) for 30 min and IκB-α
was immunoprecipitated with an anti-IκB-αantibody. Tyr-
phosphorylation of IκB-αand total-IκB-αwere then analyzed by
immunoblotting. (B) GMCs were transfected with Cx43-siRNA or
GFP-Cx43 under the condition of normal glucose (NG; 5.5 mmol/L).
After 48 h of transfection, GMCs were exposed to high glucose (HG;
30 mmol/L) for 30 min. IκB-αwas immunoprecipitated with an anti-
IκB-αantibody, and Tyr-phosphorylation of IκB-αand c-Src, and
total-IκB-αwere analyzed by immunoblotting. Experiments were
performed at least three times with similar results.
*
P<0.05 vs.
normal glucose-treated group,
#
P<0.05 vs. 30 mmol/L
glucose-treated group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 11 of 17
http://www.biosignaling.com/content/11/1/38
rebuilt GJIC inhibited by high glucose. However, Cx43CT
overexpression did not exhibit such effects. Similar to the
restoration of Cx43, Cx43CT reduced the activation of
c-Src and NF-κB in GMCs exposed to high glucose, which
suggests that this effect depends mostly on the interaction
between Cx43CT and c-Src.
Our results confirm our hypothesis that Cx43 regu-
lates the activity of c-Src in high glucose-treated GMCs
and activates NF-κB. We further investigated the effects
of Cx43 on protein expression of target genes of NF-κB,
including ICAM-1 and TGF-β1, in high glucose-treated
GMCs. ICAM-1 is an important downstream inflamma-
tory factor whose gene contains an NF-κB binding site in
the promoter region [41]. ICAM-1 gene deficiency pre-
vents nephropathy in type 2 diabetic db/db mice [26,27].
TGF-β1 is recognised as another important factor in DN
pathogenesis by mediating inflammatory responses, which
aggravates accumulation of the ECM proteins FN and col-
lagen, and interstitial myofibroblast activation, a critical
event in the pathogenesis of interstitial fibrosis [28-30].
Figure 8 Regulation of high glucose-induced NF-κB nuclear translocation and c-Src activity by Cx43 is independent of GJIC. GMCs were
transfected with GFP-Cx43 or Flag-Cx43CT under the condition of normal glucose (NG; 5.5 mmol/L). After 48 h of transfection, GMCs were
exposed to high glucose (HG; 30mmol/L) for 30 min. (A) Photomicrographs obtained after Lucifer yellow scrape-loading in GMCs transfected with
GFP-Cx43 and Flag-Cx43CT (magnification 100×, upper panel). Phase contrast views are also provided (lower panel). Scale bar represents 100 μm.
(B) Proteins were extracted for analysis of NF-κB p65 nuclear translocation by immunoblotting. Histone H1.4 and α-tubulin were measured by
immunoblotting as a loading control. (C) c-Src was immunoprecipitated with an anti-c-Src antibody and Flag, phosphorylation of Tyr416 on c-Src
(Y416-c-Src) and total c-Src were analyzed by immunoblotting. Experiments were performed at least three times with similar results.
*
P<0.05 vs.
normal glucose-treated group,
#
P<0.05 vs. 30 mmol/L glucose-treated group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 12 of 17
http://www.biosignaling.com/content/11/1/38
Figure 9 (See legend on next page.)
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 13 of 17
http://www.biosignaling.com/content/11/1/38
In the current study, restoration of Cx43 or Cx43CT re-
versed high glucose-induced increases in ICAM-1 and
TGF-β1 protein expression in GMCs. FN is an important
factor in the ECM and excessive synthesis of it contributes
to glomerular basement membrane thickening and glom-
erular sclerosis. Several studies have proposed that Cx43
may play an important role in cardiac and pulmonary fi-
brosis. Mice lacking Cx40 and endothelial cell Cx43 have
lung dysfunction and fibrosis [42]. Reduced Cx43 expres-
sion increases fibrosis and pro-arrhythmia in aged and
pressure-overloaded mice due to enhanced fibroblast ac-
tivity [43]. In our study, high glucose or silencing of Cx43
by Cx43-siRNA induced the upregulation of ICAM-1,
TGF-β1 and FN. Overexpression of Cx43 and Cx43CT at-
tenuated the increase in FN induced by high glucose in
GMCs, confirming the importance of Cx43 in renal fibro-
sis. The c-Src inhibitor PP2 also exhibited an inhibitory ef-
fect on the overexpression of ICAM-1, TGF-β1andFN
induced by high glucose, thus confirming the role of c-Src
in the activation of NF-κB.
Conclusions
Our study describes a novel mechanism of NF-κB ac-
tivation in high glucose-treated GMCs involving Cx43.
In summary, downregulation of Cx43 induced by high
glucose activates c-Src, which in turn promotes inter-
action between c-Src and IκB-αand contributes to NF-
κB activation, leading to renal inflammation. The results
presented in this study show that Cx43 induces NF-κB
activation and fibrosis in GMCs, which is beneficial for
the development of new therapies against DN. However,
the mechanism by which regulation of Cx43 expression
occurs requires further study.
Methods
Cell culture and transfection
Rat GMCs were separated from the glomeruli of Sprague–
Dawley (SD) rats and identified via a specific assay as
previously described [44]. The cultured cells were used
at confluence between the 5
th
and 8
th
passages. Con-
fluent cells were rendered quiescent by incubation for
24 h in serum-free medium before treating with glucose
(5.5 mmol/L as normal glucose and 30 mmol/L as high
glucose) or osmotic control (mannitol, 30 mmol/L final
concentration) for various times. 10 μM PP2 (c-Src in-
hibitors) or 10 μM PP3 (inactive analogue) were added
before high glucose for 30 min (Sigma-Aldrich, St. Louis,
MO). Transfection of GFP-Cx43, Flag-Cx43CT (Addgene,
Cambridge, MA) and Cx43-siRNA plasmid (gift from Tao
Liang professor, Zhongshan School of Medicine, SYSU,
China) were performed per the manufacturer’sinstruction
for Lipofectamine™LTX &Plus Reagent (Life Technolo-
gies, Carlsbad, CA).
Immunoprecipitation and immunoblotting
The cell monolayers were lysed in a cell lysis buffer for im-
munoprecipitation (Beyotime, Jiangsu, China). Immuno-
precipitation was performed by incubating 0.5 mg cell
lysate protein which was determined by bicinchoninic acid
assay (BCA) according to the manufacture’sinstructions
(Thermo Fisher Scientific, Rockford, IL) with 1μg of corre-
sponding antibody and protein G/A agarose bead (Merk,
Darmstadt, Germany) at 4°C overnight. Immunoblotting
was performed as previously described [45]. Kidney tissues
were lysed, proteins were extracted as previously published
[46]. The nuclear and cytoplasmic proteins of GMCs were
extracted using a commercially available assay kit (Active
Motif, Carlsbad, CA) and the total proteins were extracted
as published [46]. The signals were visualized by a GE
ImageQuant LAS4000mini, and analyzed using the Quan-
tity One Protein Analysis Software (Bio-Rad Laboratories,
Hercules, CA). The antibodies included mouse monoclo-
nal antibodies against connexin43, NF-κBp65,Inhibitorof
κB-α(IκB-α), p-Tyr and FN, rabbit polyclonal antibody
against c-Src, goat polyclonal anti-body against ICAM-1
and ZO-1 (Santa Cruz Biotechnology, Santa Cruz, CA),
rabbit monoclonal antibodies against phospho-c-Src (Tyr),
connexin43, phospho-IκB-α(Ser) and TGF-β(Cell Signal-
ling, Danvers, MA), rabbit monoclonal antibodies against
Histone H1.4 and α-tubulin (Sigma–Aldrich, St. Louis,
MO), mouse monoclonal antibodies against Thy-1.1 and
RECA-1 (Abcam, Cambridge, MA).
Confocal laser scanning fluorescence microscopy (LSCM)
Different groups of adherent cells were washed with
phosphate-buffered saline (PBS), fixed with 4% parafor-
maldehyde in PBS for 20 min, and permeabilized with
0.1% TritonX-100 for 5 min at room temperature. Cells
(See figure on previous page.)
Figure 9 Cx43 inhibits high glucose-induced expression of FN, ICAM-1, and TGF-β1. (A) GMCs were transfected with GFP-Cx43 or Flag-
Cx43CT in normal glucose (NG; 5.5 mmol/L). After 48 h of transfection, GMCs were exposed to high glucose (HG; 30 mmol/L) for 24 h. Proteins
were extracted for analysis of FN, ICAM-1, and TGF-β1 by immunoblotting. (B) GMCs were transfected with Cx43-siRNA in normal glucose. After
24 h, cells were then transfected with GFP-Cx43 to restore Cx43 expression. Proteins were extracted for analysis of FN, ICAM-1, and TGF-β1by
immunoblotting. The high glucose-treated group was used as the control. (C) Immunofluorescence images stained doubly with anti-Cx43
antibody and anti-FN antibody were captured under a laser scanning confocal microscope (magnification 630×). Green fluorescence indicates
Cx43. Red fluorescence indicates FN. Blue fluorescence indicates nuclei. Scale bar represents 20 μm. Experiments were performed at least three
times with similar results.
*
P<0.05 vs. normal glucose-treated group,
#
P<0.05 vs. 30 mmol/L glucose-treated group.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 14 of 17
http://www.biosignaling.com/content/11/1/38
were incubated with antibodies against NF-κB p65,
connexin43, c-Src, IκB-αor FN overnight at 4°C after
blocking with 10% goat serum. Or frozen kidney sec-
tions (7.5 μm) were incubated with antibodies against
Cx43, Thy-1.1 and RECA-1 overnight at 4°C after
blocking with 10% goat serum. Then the cells and sec-
tions were incubated in the dark at room temperature
for 1 h with a secondary antibody (Alexa Fluor® 488,
Alexa Fluor® 546, Invitrogen, Carlsbad, CA). The nu-
cleus was stained with Hoechst33342. Cells and sections
were placed under a laser scanning confocal microscope
(LSM710, Carl Zeiss, Germany) for observation and
image acquisition.
Assessment of gap junctional intercellular communication
Gap junction permeability was determined by the scrape-
loading/dye transfer technique [37]. Scrape-loading was
performed by scraping the cell layer with a broken razor
blade in culture media containing Lucifer yellow (1 mg/
ml, Life Technologies, Carlsbad, CA). Lucifer yellow is a
low molecular weight (457 Da) fluorescent dye that can
pass through the gap junctions of loaded cells to their
neighbors. After 2 min, the dye solution was removed and
the cells were carefully washed. Subsequently, 5 min after
scraping, fluorescence photomicrographs were captured
with a laser scanning confocal microscope (LSM710, Carl
Zeiss, Germany). At least six photomicrographs of the
centre of the dish were taken and the fluorescent area oc-
cupied by Lucifer yellow in the images was measured
with the image-analyzer software (Scion Image, Scion
Corporation, Frederick, MD).
Animal experiment
Male SD rats (n=20, 200±10 g) were obtained from
the Laboratory Animal Center, Sun Yat-sen Univer-
sity, Guangzhou, China Animal (Quality Certificate No.:
0005201). db/db (male, n=10, 40±5 g) mice were obtained
from the model animal research center of Nanjing Univer-
sity (Quality Certificate No.: 0007963). All animal proce-
dures conformed to the China Animal Welfare Legislation
and were reviewed and approved by the Sun Yat-sen
University Committee on Ethics in the Care and Use of
Laboratory Animals. All animals were housed under stand-
ard conditions with free access to regular food and water.
After feeding with regular diet for 1 week, STZ-diabetic
rats were induced as previously reported [24]. Diabetic
rats were confirmed by the levels of fasting blood glu-
cose measurement (≥16.7 mmol/l after 72 h injection).
It was continued for 12 weeks, after which the rats were
sacrificed. db/db mice were sacrificed at the time when
they were 12 weeks age. Kidney samples were rapidly
excised, weighed and frozen in liquid nitrogen and then
stored at −80°C or fixed in 10% neutral-buffered formalin.
Immunohistochemistry
Kidney sections 4 μm thick were processed using a stand-
ard immunostaining protocol as previously reported [47].
A negative control was prepared by omitting the primary
antibody.
Statistical analysis
All experiments were performed at least in triplicate.
The data were assessed using SPSS 11.5. All values were
expressed as mean ± SD. Statistical analyses of data were
performed by one-way ANOVA using post-hoc multiple
comparisons. P< 0.05 was considered to be statistically
significant.
Additional files
Additional file 1: Figure S1. Half-life of Cx43 was explored in GMCs
cultured in normal glucose or high glucose using cycloheximide.
(A) GMCs cultured in normal glucose (5.5 mM) were co-incubated with
10 μM cycloheximide for the indicated time. (B) GMCs cultured in high
glucose (30 mM) were co-incubated with 10 μM cycloheximide for
the indicated time. (C) GMCs were treated for 30 min with increasing
concentrations of glucose as indicated. Then proteins were extracted for
analysis of Cx43 by immunoblotting. α-Tubulin was used as a loading
control.
*
P<0.05 vs. control group. Chx, cycloheximide.
Additional file 2: Figure S2. HIF-1αis regulated in the GMCs by high
glucose or low levels of Cx43. (Aand B) GMCs were treated with high
glucose for the indicated time, then proteins were extracted for analysis of
HIF-1αby immunoblotting. (C) GMCs were transfected with Cx43-siRNA in
normal glucose (5.5 mM) or GFP-Cx43 in high glucose (30 mM). After 48 h,
proteins were extracted for analysis of Cx43 and HIF-1αby immunoblotting.
(D) GMCs cultured in high glucose were co-incubated with PP2 or PP3
(10 μM) or PDTC (100 μM). After 48 h, proteins were extracted for analysis
of HIF-1αby immunoblotting. α-Tubulin was used as a loading control.
*
P<0.05 vs. normal glucose-treated group.
#
P<0.05 vs. high glucose-treated
group.
Abbreviations
Cx43: Connexin43; Cx43CT: Carboxy tail of Cx43; DN: Diabetic nephropathy;
ECM: Extracellular matrix; FN: Fibronectin; GJIC: Gap junctional intercellular
communication; GMCs: Glomerular mesangial cells; ICAM-1: Intercellular
adhesionmolecule-1; IKK: IκB kinase; IκB-α: Inhibitor of κB-α; NF-κB: Nuclear
factor kappa-B; TGF-β1: Transforming growth factor-beta 1; SD rats: Sprague–
Dawley rats.
Competing interest
The authors declare that there is no conflict of interest associated with this
manuscript.
Authors’contributions
XX designed and performed experiments, acquisition and analysis of data,
and drafted the manuscript. TL, XTC and KPH helped to perform experiments
and prepare the manuscript. JH, SGW and CC have conceived of the study,
participated in its design and coordination. HQH, PQL and XYS have been
involved in drafting the manuscript and revising it critically for important
intellectual content. All authors have read and approved the final version of
this manuscript.
Acknowledgement
This study was supported by research grants from the National Natural
Science Foundation of China (No. 81170676), the Science, Technology
Program of Guangdong province, PR China (No.2011A080502004) and
National Natural Science Foundation of Guangdong province (No.
S2012020010991). The authors appreciate Dr. Liang Tao (Zhongshan School
of Medicine, SYSU, China) for providing Cx43-siRNA plasmid.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 15 of 17
http://www.biosignaling.com/content/11/1/38
Author details
1
Laboratory of Pharmacology & Toxicology, School of Pharmaceutical
Sciences, Sun Yat-sen University, Guangzhou 510006, China.
2
Department of
Pharmaceutical Engineering, Ocean College, Hainan University, Haikou
570228, China.
3
Vascular Biology Institute, Guangdong Pharmaceutical
University, Guangzhou 510006, China.
Received: 8 January 2013 Accepted: 10 May 2013
Published: 29 May 2013
References
1. Mason RM, Wahab NA: Extracellular matrix metabolism in diabetic
nephropathy. J Am Soc Nephrol 2003, 14:1358–1373.
2. Mariappan MM: Signaling mechanisms in the regulation of renal matrix
metabolism in diabetes. Exp Diabetes Res 2012, 2012:749812.
3. Tuttle KR: Linking metabolism and immunology: diabetic nephropathy is
an inflammatory disease. J Am Soc Nephrol 2005, 16:1537–1538.
4. Navarro-Gonzalez JF, Mora-Fernandez C, de Fuentes MM, Garcia-Perez J:
Inflammatory molecules and pathways in the pathogenesis of diabetic
nephropathy. Nat Rev Nephrol 2011, 7:327–340.
5. Bondar IA, Klimontov VV, Nadeev AP: Urinary excretion of
proinflammatory cytokines and transforming growth factor beta at early
stages of diabetic nephropathy. Ter Arkh 2008, 80:52–56.
6. Iwamoto M, Mizuiri S, Arita M, Hemmi H: Nuclear factor-kappa B activation
in diabetic rat kidney: evidence for involvement of P-selectin in diabetic
nephropathy. Tohoku J Exp Med 2005, 206:163–171.
7. Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, Schneider H,
Ruiz-Ortega M, Egido J: NF-kappa B activation and overexpression of
regulated genes in human diabetic nephropathy. Nephrol Dial Transplant
2004, 19:2505–2512.
8. Herve JC, Bourmeyster N, Sarrouilhe D, Duffy HS: Gap junctional complexes:
from partners to functions. Prog Biophys Mol Biol 2007, 94:29–65.
9. Evans WH, Martin PE: Gap junctions: structure and function (Review). Mol
Membr Biol 2002, 19:121–136.
10. Yao J, Zhu Y, Morioka T, Oite T, Kitamura M: Pathophysiological roles of gap
junction in glomerular mesangial cells. JMembrBiol2007, 217:123–130.
11. Zhang X, Chen X, Wu D, Liu W, Wang J, Feng Z, Cai G, Fu B, Hong Q, Du J:
Downregulation of connexin 43 expression by high glucose induces
senescence in glomerular mesangial cells. J Am Soc Nephrol 2006,
17:1532–1542.
12. Sawai K, Mukoyama M, Mori K, Yokoi H, Koshikawa M, Yoshioka T, Takeda R,
Sugawara A, Kuwahara T, Saleem MA, et al:Redistribution of connexin43
expression in glomerular podocytes predicts poor renal prognosis in
patients with type 2 diabetes and overt nephropathy. Nephrol Dial
Transplant 2006, 21:2472–2477.
13. Takenaka T, Inoue T, Okada H, Ohno Y, Miyazaki T, Chaston DJ, Hill CE,
Suzuki H: Altered gap junctional communication and renal
haemodynamics in Zucker fatty rat model of type 2 diabetes.
Diabetologia 2011, 54:2192–2201.
14. Liu L, Hu X, Cai GY, Lv Y, Zhuo L, Gao JJ, Cui SY, Feng Z, Fu B, Chen XM:
High glucose-induced hypertrophy of mesangial cells is reversed by
connexin43 overexpression via PTEN/Akt/mTOR signaling. Nephrol Dial
Transplant 2012, 27:90–100.
15. Giepmans BN: Role of connexin43-interacting proteins at gap junctions.
Adv Cardiol 2006, 42:41–56.
16. Giepmans BN, Hengeveld T, Postma FR, Moolenaar WH: Interaction of c-Src
with gap junction protein connexin-43: role in the regulation of cell-cell
communication. J Biol Chem 2001, 276:8544–8549.
17. Gilleron J, Fiorini C, Carette D, Avondet C, Falk MM, Segretain D, Pointis G:
Molecular reorganization of Cx43, Zo-1 and Src complexes during the
endocytosis of gap junction plaques in response to a non-genomic
carcinogen. J Cell Sci 2008, 121:4069–4078.
18. Suzaki Y, Yoshizumi M, Kagami S, Nishiyama A, Ozawa Y, Kyaw M, Izawa Y,
Kanematsu Y, Tsuchiya K, Tamaki T: BMK1 is activated in glomeruli of
diabetic rats and in mesangial cells by high glucose conditions. Kidney
Int 2004, 65:1749–1760.
19. Mima A, Abe H, Nagai K, Arai H, Matsubara T, Araki M, Torikoshi K, Tominaga
T, Iehara N, Fukatsu A, et al:Activation of Src mediates PDGF-induced
Smad1 phosphorylation and contributes to the progression of
glomerulosclerosis in glomerulonephritis. PLoS One 2011, 6:e17929.
20. Alonso F, Krattinger N, Mazzolai L, Simon A, Waeber G, Meda P, Haefliger JA:
An angiotensin II- and NF-kappa B-dependent mechanism increases
connexin 43 in murine arteries targeted by renin-dependent
hypertension. Cardiovasc Res 2010, 87:166–176.
21. Zhao Y, Rivieccio MA, Lutz S, Scemes E, Brosnan CF: The TLR3 ligand polyI :
C downregulates connexin43 expression and function in astrocytes by a
mechanism involving the NF-kappa B and P13 kinase pathways. Glia
2006, 54:775–785.
22. Huang WC, Chen JJ, Chen CC: c-Src-dependent tyrosine phosphorylation of
IKK beta is involved in tumor necrosis factor-alpha-induced intercellular
adhesion molecule-1 expression. J Biol Chem 2003, 278:9944–
9952.
23. Fan CG, Li Q, Ross D, Engelhardt JF: Tyrosine phosphorylation of I kappa B
alpha activates NF kappa B through a redox-regulated and c-Src
-dependent mechanism following hypoxia/reoxygenation. J Biol Chem
2003, 278:2072–2080.
24. Xie X, Peng J, Huang K, Huang J, Shen X, Liu P, Huang H: Polydatin
ameliorates experimental diabetes-induced fibronectin through
inhibiting the activation of NF-kappaB signaling pathway in rat
glomerular mesangial cells. Mol Cell Endocrinol 2012, 362:183–193.
25. Singh D, Solan JL, Taffet SM, Javier R, Lampe PD: Connexin 43 interacts
with zona occludens-1 and −2 proteins in a cell cycle stage-specific
manner. J Biol Chem 2005, 280:30416–30421.
26. Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH: Intercellular
adhesion molecule-1 deficiency is protective against nephropathy in
type 2 diabetic db/db mice. J Am Soc Nephrol 2005, 16:1711–1722.
27. Okada S, Shikata K, Matsuda M, Ogawa D, Usui H, Kido Y, Nagase R, Wada J,
Shikata Y, Makino H: Intercellular adhesion molecule-1-deficient mice are
resistant against renal injury after induction of diabetes. Diabetes 2003,
52:2586–2593.
28. Sharma K, Jin Y, Guo J, Ziyadeh FN: Neutralization of TGF-beta by anti-TGF
-beta antibody attenuates kidney hypertrophy and the enhanced
extracellular matrix gene expression in STZ-induced diabetic mice.
Diabetes 1996, 45:522–530.
29. Murphy M, Docherty NG, Griffin B, Howlin J, McArdle E, McMahon R, Schmid
H, Kretzler M, Droguett A, Mezzano S, et al:IHG-1 amplifies TGF-beta1
signaling and is increased in renal fibrosis. J Am Soc Nephrol 2008,
19:1672–1680.
30. Wang A, Ziyadeh FN, Lee EY, Pyagay PE, Sung SH, Sheardown SA, Laping
NJ, Chen S: Interference with TGF-beta signaling by Smad3-knockout in
mice limits diabetic glomerulosclerosis without affecting albuminuria.
Am J Physiol Renal Physiol 2007, 293:F1657–1665.
31. Musil LS, Goodenough DA: Biochemical analysis of connexin43
intracellular transport, phosphorylation, and assembly into gap
junctional plaques. J Cell Biol 1991, 115:1357–1374.
32. Laird DW, Puranam KL, Revel JP: Turnover and phosphorylation dynamics
of connexin43 gap junction protein in cultured cardiac myocytes.
Biochem J 1991, 273(Pt 1):67–72.
33. Laird DW, Castillo M, Kasprzak L: Gap junction turnover, intracellular
trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat
mammary tumor cells. J Cell Biol 1995, 131:1193–1203.
34. Schaeffer G, Levak-Frank S, Spitaler MM, Fleischhacker E, Esenabhalu VE,
Wagner AH, Hecker M, Graier WF: Intercellular signalling within vascular
cells under high D-glucose involves free radical-triggered tyrosine kinase
activation. Diabetologia 2003, 46:773–783.
35. Huang Q, Sheibani N: High glucose promotes retinal endothelial cell
migration through activation of Src, PI3K/Akt1/eNOS, and ERKs. Am J
Physiol Cell Physiol 2008, 295:C1647–1657.
36. Lau AF: c-Src: bridging the gap between phosphorylation- and
acidification-induced gap junction channel closure. Sci STKE 2005, 2005:
pe33.
37. Gangoso E, Herrero-Gonzalez S, Giaume C, Naus C, Medina JM, Tabernero A:
Connexin43 inhibits the oncogenic activity of c-Src in C6 glioma cells.
FEBS J 2011, 278:456–456.
38. Valle-Casuso JC, Gonzalez-Sanchez A, Medina JM, Tabernero A: HIF-1 and
c-Src mediate increased glucose uptake induced by endothelin-1 and
Connexin43 in astrocytes. PLoS One 2012, 7:e32448.
39. Kumar A, Takada Y, Boriek AM, Aggarwal BB: Nuclear factor-kappa B: its role
in health and disease. Journal of Molecular Medicine-Jmm 2004, 82:434–448.
40. Bui NT, Livolsi A, Peyron JF, Prehn JHM: Activation of nuclear factor kappa
B and bcl-x survival gene expression by nerve growth factor requires
tyrosine phosphorylation of I kappa B alpha. J Cell Biol 2001, 152:753–763.
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 16 of 17
http://www.biosignaling.com/content/11/1/38
41. Bunting K, Rao S, Hardy K, Woltring D, Denyer GS, Wang J, Gerondakis S,
Shannon MF: Genome-wide analysis of gene expression in T cells to
identify targets of the NF-kappa B transcription factor c-Rel. J Immunol
2007, 178:7097–7109.
42. Koval M, Billaud M, Straub AC, Johnstone SR, Zarbock A, Duling BR, Isakson
BE: Spontaneous lung dysfunction and fibrosis in mice lacking connexin
40 and endothelial cell connexin 43. Am J Pathol 2011, 178:2536–2546.
43. Jansen JA, van Veen TA, de Jong S, van der Nagel R, Driessen HE, Labzowski
RP, Bosch AA, Oefner CM, Vos MA, de Bakker JM, van Rijen HV: Reduced
Cx43 expression leads to increased fibrosis and Pro-arrhythmia due to
enhanced fibroblast activity in aged and pressure overloaded mice.
Circulation 2010, 122:A19348.
44. Geoffroy K, Wiernsperger N, Lagarde M, El Bawab S: Bimodal effect of
advanced glycation end products on mesangial cell proliferation is
mediated by neutral ceramidase regulation and endogenous
sphingolipids. J Biol Chem 2004, 279:34343–34352.
45. Jiang Q, Liu PQ, Wu XQ, Liu WH, Shen XY, Lan TA, Xu SW, Peng J, Xie X,
Huang HQ: Berberine attenuates lipopolysaccharide-induced extracelluar
matrix accumulation and inflammation in rat mesangial cells:
Involvement of NF-kappa B signaling pathway. Mol Cell Endocrinol 2011,
331:34–40.
46. Liu WH, Zhang XY, Liu PQ, Shen XY, Lan TA, Li WY, Jiang Q, Xie X, Huang
HQ: Effects of berberine on matrix accumulation and NF-kappa B signal
pathway in alloxan-induced diabetic mice with renal injury. Eur J
Pharmacol 2010, 638:150–155.
47. Lan T, Shen X, Liu P, Liu W, Xu S, Xie X, Jiang Q, Li W, Huang H: Berberine
ameliorates renal injury in diabetic C57BL/6 mice: involvement of
suppression of SphK-S1P signaling pathway. Arch Biochem Biophys 2010,
502:112–120.
doi:10.1186/1478-811X-11-38
Cite this article as: Xie et al.:Connexin43 mediates NF-κB signalling
activation induced by high glucose in GMCs: involvement of c-Src. Cell
Communication and Signaling 2013 11:38.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Xie et al. Cell Communication and Signaling 2013, 11:38 Page 17 of 17
http://www.biosignaling.com/content/11/1/38
