Tumor necrosis factor-alpha inhibits Schwann cell proliferation by up-regulating Src-suppressed protein kinase C substrate expression

, ,1 ,1

*Laboratory Center, Affiliated Hospital of Nantong University, and The Jiangsu Province Key Laboratory of Neuroregeneration,

Nantong University, Nantong, China

�Department of Immunology, Medical College, Nantong University, Nantong, China

Schwann cells (SCs), the predominant glial cells of the PNS,are responsible for the protection and support of axons, andthe synthesis of myelin sheaths. During development, SCsproliferate and wrap around selected axons leading to theformation of myelin. Following injury, SCs dedifferentiate,accompanied by a wave of proliferation (Fernandez-Valleet al. 1995). If axon regrowth is allowed, SCs stop dividingand start remyelination (Walikonis and Poduslo 1998).Recent studies showed that cyclin D1 is functionallyessential in mature SCs in a repair setting. SCs fail toproliferate in cyclin D1-null mice during Wallerian degen-eration (Kim et al. 2000; Atanasoski et al. 2001). Further-more, SCs from cyclin D1)/) animals do not respond togrowth factors such as neuregulin and platelet-derivedgrowth factor (Kim et al. 2000).

Tumor necrosis factor-alpha (TNF-a) is a primary medi-ator of inflammatory responses and is mainly synthesized andreleased by SCs in the PNS after injury (Wagner and Myers1996). Several lines of evidence suggest that TNF-a, which

is released into the local milieu after injury, might initiate thecascade that triggers Wallerian degeneration and inhibit theproliferation of SCs in a dose-dependent manner (Chandrosset al. 1996). However, the mechanisms by which TNF-ainduces SC growth arrest have not been well revealed.Connexin46 and gap junctional communication as well asNGFRp75 signaling mechanisms are reported to be involvedin TNF-a-induced SC growth arrest (Chandross et al. 1996;

Received April 7, 2009; revised manuscript received/accepted July 23,2009.Address correspondence and reprint requests to Aiguo Shen and

Huming Wang, Laboratory Center, Affiliated Hospital of Nantong Uni-versity, and The Jiangsu Province Key Laboratory of Neuroregeneration,Nantong University, 19 Qixiu Road, Nantong 226001, China.E-mail: [email protected]; [email protected] authors contributed equally to this study.Abbreviations used: CDK, cyclin dependent kinase; ERK1/2, extra-

cellular signal-regulated kinase 1/2; PKC, protein kinase C; SC, Schw-ann cell; SSeCKS, Src-suppressed protein kinase C substrate; TNF-a,tumor necrosis factor-alpha.

Abstract

Src-suppressed protein kinase C substrate (SSeCKS) is a

protein kinase C substrate protein, which plays an important

role in mitogenic regulatory activity. In the early stage of nerve

injury, expression of SSeCKS in the PNS increases, mainly in

Schwann cells (SCs). However, the exact function of SSeCKS

in the regulation of SC proliferation remains unclear. In this

study, we found that tumor necrosis factor-alpha (TNF-a) in-

duced both SSeCKS a isoform expression and SC growth

arrest in a dose-dependent manner. By knocking down

SSeCKS a isoform expression, TNF-a-induced growth arrest

in SCs was partially rescued. Concurrently, the expression of

cyclin D1 was reduced and the activity of extracellular signal-

regulated kinase 1/2 was decreased. A luciferase activity as-

say showed that cyclin D1 expression was regulated by

SSeCKS at the transcription level. In addition, the cell frag-

ments assay and immunofluorescence revealed that TNF-a

prevented the translocation of cyclin D1 into the nucleus, while

knocking down SSeCKS a isoform expression prompted cy-

clin D1 redistribution to the nucleus. In summary, our data

indicate that SSeCKS may play a critical role in TNF-a-in-

duced SC growth arrest through inhibition of cyclin D1

expression thus preventing its nuclear translocation.

Keywords: cyclin D1, extracellular signal-regulated kinase 1/

2, Schwann cell, SSeCKS, tumor necrosis factor-alpha.

J. Neurochem. (2009) 111, 647–655.

JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 647–655 doi: 10.1111/j.1471-4159.2009.06346.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 647–655 647

Boyle et al. 2005). In oligodendroglial lineage cells, TNF-acan suppress cyclin D1 expression and thus reduce cellproliferation (Yu et al. 2000). Nevertheless, the criticaltargets of cyclin D1 in TNF-a-induced SC growth arreststill remain yet to be unidentified.

Protein kinase C (PKC) triggers cellular signals thatregulate proliferation or survival in cell- and stimulus-specific manner. Activation of PKC prevents TNF-a-inducedcell growth arrest in both C6 glioma cells and mousefibroblasts L929 cells (Lung et al. 2005; Byun et al. 2006).PKC activity, especially PKC-a activity is involved in theregulation of SC proliferation (Kamiya et al. 2003), but thedownstream signaling pathways involved still remainunknown. One of the most prominent intracellular substratesof PKC is Src-suppressed protein kinase C substrate(SSeCKS), which has been investigated in several cell typesincluding SCs (Lin et al. 1996; Streb et al. 2004; Chen et al.2008). SSeCKS was originally identified in a screen forgenes severely down-regulated by v-Src (Lin et al. 1995),and characterized as a major substrate of PKC both in vitroand in vivo (Lin et al. 1996). The SSeCKS gene locus inhuman and rodents encodes three major transcripts under thecontrol of three independent promoters, designated a, b, andc that are separated by 84 kbp (Streb et al. 2004). Two majorprotein isoforms of SSeCKS, a and b, are expressedubiquitously in most cell and tissue types (Gelman 2002),whereas the expression of the c isoform is restricted to thetestes (Camus et al. 2001). In addition to PKC, SSeCKS alsobinds with protein kinase A, calmodulin, cyclin D1, b-adrenergic receptor, b-1,4 galactosyltransferase, and F-actin(Gelman 2002). Together with its ability to scaffold keysignaling and cytoskeletal proteins, SSeCKS plays importantroles in regulating G1/S phase transition (Lin et al. 2000).The expression of AKAP12, the human orthologue ofSSeCKS, is down-regulated by several oncogenes in varioushuman epithelial tumors including prostate, breast, ovaries,gastric and lungs (Perou et al. 2000; Dhanasekaran et al.2001; Okuda et al. 2001). It has been documented that theserine phosphorylation of SSeCKS is induced during late G1phase by either serum growth factors or activated v-Src (Liuet al. 2006). Over-expression of AKAP12 decreases thecyclin D1 level, and prevents cyclin D1 translocation fromplasma to nucleus (Yoon et al. 2007). Recent studiesconfirmed that the expression of SSeCKS protein reachesits peak level during the early stages of peripheral nerveinjury, in parallel with TNF-a expression in SCs (Wagner andMyers 1996; Chen et al. 2008). Our group also demonstratedthat TNF-a could up-regulate SSeCKS expression in astro-cytes (Sun et al. 2007b), but whether or not TNF-ainfluences SSeCKS expression in SCs has not yet beendetermined.

In this study, we showed that TNF-a potently inhibited SCproliferation by up-regulating the expression of the SSeCKSa isoform, which in turns reduced cyclin D1 expression via

suppression of the activation of extracellular signal-regulatedkinase 1/2 (ERK1/2). SSeCKS a isoform also preventedtranslocation of cyclin D1 from cytoplasm to nucleus. Thesedata indicate that SSeCKS plays an essential role in TNF-a-induced SC growth arrest.

Materials and methods

Schwann cells cultureIsolation and culture of primary SCs was described previously

(Brockes et al. 1979). In brief, sciatic nerves were dissected out

from 1- to 3-day-old Sprague–Dawley rat pups. They were shredded

with fine forceps in D-Hank’s solution containing 0.03% collage-

nase A and 0.25% trypsin, then incubated at 37�C for 30 min, with

occasionally mixing. The dissociated cells were grown in

Dulbecco’s modified essential medium supplemented with 10%

heat-inactivated fetal bovine serum (FBS). Non-SCs were elimi-

nated as follows: the cells were incubated for 3 days in the presence

of 0.01 mM Ara-C and then grown for 4 days in complete

Dulbecco’s modified essential medium with 2 lM forskolin to

stimulate SC proliferation (Porter et al. 1986). Cells were subcultureevery 4–7 days. For all experiments, the cells were subculture no

more than five times and the cells were maintained in vitro for no

longer than 6 weeks. SCs were withdrawn from mitogen stimulation

for 1 day prior to the assays. In the condition, these cells were still

proliferation (Appendix S1, Fig. S1, Table S1).

Statistical analysisAll experiments were repeated at least three times. All numerical

data were described as mean ± SD. Data were analyzed using the

two-tailed t-test. A probability value of 0.05 or less was considered

significant. In some cases, SD values were too small to appear in the

graph as error bars.

Results

TNF-a inhibits SC proliferation, but does not induce SCapoptosisSchwann cells were treated with TNF-a at different concen-trations (0, 0.01, 0.1, 1 and 10 ng/mL) for 12 h. 1 ng/mL and10 ng/mL TNF-a decreased the proliferation rate over 50%and 75%, respectively, as compared to the control(Fig. 1a).However, the number of treated SCs was not belowthe initial seeded numbers. At the same time, we also usedflow-cytometry to investigate whether TNF-a caused SCcycle arrest. We found that TNF-a at 10 ng/mL induced cellcycle arrest in the G0/G1 phase rather than the change of thenumber of apoptotic cells (Table 1). Tunnel assay alsoconfirmed that there were no apoptotic cells after TNF-atreatment (data not shown). Western blot analysis for cleavedcaspase 3 expression showed no significant differencebetween TNF-a-treated or untreated SCs (Fig. 1b). Theseresults suggested that TNF-a inhibited SC proliferation in adose-dependent manner without inducing SCs apoptosis.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 647–655� 2009 The Authors

648 | T. Tao et al.

TNF-a up-regulates SSeCKS mRNA and protein levels inSCs along with a similar increase in SSeCKSphosphorylationRT-PCR results revealed that TNF-a induced SSeCKSmRNA expression in a dose-dependent manner. Aftertreatment with 0.1–10 ng/mL TNF-a for 12 h, SSeCKSmRNAwas gradually increased (Fig. 2a). PCR analysis withisoform-specific primers showed that the increase in SSeCKSmRNA expression was limited to the a isoform. The increaseof SSeCKS protein profile after TNF-a treatment for 12 hparalleled with mRNA and only the 290 kDa a isoform ofSSeCKS was gradually increased whereas the 280 kDa bisoform was not significantly changed (Fig. 2b). Theseresults prompted us to focus on the function of the a isoformof SSeCSK in the regulation of the SC proliferationfollowing TNF-a treatment. SSeCKS binds PKC in aphosphatidylserine-dependent manner and it is also a majorPKC substrate in vitro and in vivo (Gelman et al. 1998). Wesought to evaluate the phosphorylation of SSeCKS after

TNF-a treatment. Treatment of SCs with increasing concen-tration of TNF-a lead to dose-dependent increase in aisoform of SSeCKS phosphorylation (Fig. 2c). These resultsencouraged us to focus on the function of the a isoform ofSSeCSK in the regulation of the SC proliferation followingTNF-a treatment.

SSeCKS-knockdown reverses the growth arrest of SCsinduced by TNF-aTo further assess the importance of SSeCKS a isoform in SCproliferation, we transfected SCs with specific a isoformSSeCKS siRNA or a non-specific siRNAvector. As predicted,SSeCKS a isoform mRNA and protein expression wereconsiderably decreased in SSeCKS siRNA-transfected SCs.Subsequently we analyzed SSeCKS a isoform proteinexpression in the cells transfected with the siRNA aftertreatment with TNF-a for 12 h. As expected, the expression ofSSeCKS a isoform in mRNA and protein levels weredecreased compared with the untreated cells, while therewas no effect on the expression of b isoform at the mRNA orprotein levels (Fig. 3a). Although only 80% knocked downbecause of the limitation of transient transfection, theprofound reduction in the SSeCKS protein level that occurredwas indeed resulted from SSeCKS siRNA but not from theoverall inhibition of protein synthesis, as glyceraldehydephosphate dehydrogenase (GAPDH) protein level remainedunchanged across samples. After treating SSeCKS-knock-down SCs with TNF-a for 12 h, the proliferation rate showedabout 2-folds increase relative to the non-specific siRNAtransfected SCs. However this was not the case for the cellswithout TNF-a treatment (Fig. 3b). Flow cytometry analysisshowed that SSeCKS-knockdown SCs treated with 10 ng/mLTNF-a reversed cell cycle arrest in the G0/G1 phase (Table 1).

SSeCKS down-regulates cyclin D1 expression inTNF-a-treated SCsCyclin D1 is a member of the cyclin family and also aregulator of members of cyclin dependent kinases (CDK)

(a) (b)

Fig. 1 TNF-a inhibited SC proliferation, but did not cause SC

apoptosis. (a) Cells were incubated with various concentrations (0,

0.01, 0.1, 1, 10 ng/mL) of TNF-a for 12 h and 100 lM Brdu labeling

solution was added. After 4-h incubation, the anti-Brdu-POD working

solution and substrate solution were then added, to each well.

Absorbance was measured and results were calculated as fold in-

crease against the control (0 ng/mL). The data were means ± SEM.

Statistical differences compared with the controls were given as

*p < 0.05, **p < 0.01. (b) Western blot analysis determined the

expression of caspase 3 after various concentrations of TNF-a

treated. The equal amounts of precursor caspase 3 were detected,

but not the cleaved caspase 3 which is about 20 kDa. Cell lysate

from TNF-a (10 ng/mL) treated Hep G2 cells was used as a positive

apoptosis control.

Table 1 Cell-cycle profile of SSeCKS-siRNA SCs after TNF-a treat-

ment

G0/G1 S G2 Apoptosis

Control 52.9 33.7 13.4 0

Non-specific siRNA 53.8 34.2 12 0

SSeCKS-siRNA 51.8 33.3 14.9 0

TNF-a 72.2 16.8 11.0 0

TNF-a + non-specific siRNA 70.3* 17.5* 12.2 0

TNF-a + SSeCKS siRNA 49.8 32.3 17.9 0

All values are given in percentages. SCs were transfected with non-

specific siRNA or SSeCKS-siRNA vector before treated by TNF-a for

12 h. Flow cytometry of PI-stained cells were used to analysis the cell-

cycle distribution. In SC treatment with TNF-a for 12 h, more SCs were

prevented in the G1-phase. In cells transfected with the SSeCKS-

siRNA vector, G1-phase cells were decreased and more cells entered

the cell cycle after TNF-a treated. Statistical differences compared

with the controls are given as *p < 0.05.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 647–655

SSeCKS in Schwann cell proliferation | 649

(a)

(b)

(c)

Fig. 2 TNF-a up-regulated SSeCKS expression, accompany by a

similar increase in phosphorylation in SCs. (a) Total mRNA were got

from concentrations TNF-a-treated SCs using Trizol regent, mRNA

RT-PCR products amplified by the isoform specific primers or the total

SSeCKS primer stained with ethidium bromide and separated in a 1%

agarose gel. (b) Western blot analysis determined the expression of

SSeCKS protein after concentrations of TNF-a treated. (c) Cell lysates

were subjected to immunoprecipitate with SSeCKS antibody, and

blotted by anti-phosphorylated-(Ser) PKC substrate antibody (p-Ser)

to detect the phosphorylation of SSeCKS in TNF-a-treated SCs. Gels

and immunoblots were quantified and the average pixel intensity for

each band relative to control was calculated as a fold increase against

the control (0 ng/mL group), corrected by GAPDH (a, b) or SSeCKS

(c) which was used as an internal control. The data were

means ± SEM. Statistical differences compared with the controls were

given as *p < 0.05.

(a) (b)

Fig. 3 Knockdown SSeCKS a isoform expression efficiency re-

versed SC growth arrest induced by TNF-a. (a) SCs were trans-

fected with specific a isoform specific SSeCKS-siRNA vector or the

non-specific-siRNA vector for 48 h, and then treated by TNF-a

(10 ng/mL) for 12 h. (a) mRNA RT-PCR products amplified by the

isoform specific primers or the total SSeCKS primer were stained

with ethidium bromide and separated in a 1% agarose gel. (b)

Expression of SSeCKS protein determined by western blot analysis.

SSeCKS a isoform siRNA was abundant in rat SCs. (b) Non-spe-

cific siRNA and SSeCKS knock-down SCs were cultured in the

absence or presence of 10 ng/mL TNF-a for 12 h. Brdu labeling

solution was added to the medium and incubated for additional 4 h.

Absorbance was measured and calculated comparing to the non-

specific siRNA-treated SCs without TNF-a treatment. The data were

means ± SEM. Statistical differences compared with the SCs with-

out treatment were given as *p < 0.05, and after TNF-a treated,

differences compared with the TNF-a treated un-transfected SCs

were given as #p < 0.05.


650 | T. Tao et al.

that play a pivotal role in controlling progression through thecell cycle (Pines 1991). Immunoblotting experiments re-vealed that 0.1–10 ng/mL TNF-a gradually decreased thecyclin D1 expression in SCs (Fig. 4a). The effect of TNF-aon cyclin D1 expression was recovered in SSeCKS-knock-down SCs (Fig. 4c). To further determine whether or not theregulation of cyclin D1 expression by SSeCKS occurred atthe transcription level, we used a cyclin D1 promoter-luciferase reporter construct. After TNF-a treatment for 12 h,we observed that luciferase activity was decreased to 20% ofthe control. Furthermore, in SSeCKS-knockdown SCs,luciferase activity was only 20% lower than that of thecontrol while no differences were observed in the untreatedcells (Fig. 4d). These results indicated that after TNF-atreatment, SSeCKS down-regulated cyclin D1 expression atthe transcription level. ERK1/2 activation is also importantlyfor cyclin D1 expression. When U0126, the inhibitor ofERK1/2 was used, the cyclin D1 expression was stronglydecreased (Fig. 4b). Previous studies have determined thatSSeCKS most likely prevents the induction of cyclin D1

expression by inhabiting sustained activation of ERK1/2 (Linet al. 2000). Our study showed that knockdown of SSeCKSexpression could increase ERK1/2 phosphorylation inTNF-a-treated SCs (Fig. 4c).

SSeCKS prevents nuclear localization of cyclin D1 in TNF-atreated SCsThe association of cyclin D1 and SSeCKS in SCs was furtherexamined by immunoprecipitation. Cyclin D1 was found toco-immunoprecipitate with SSeCKS in normal or TNF-a-treated SCs. Conversely, immunoblot analysis of cyclin D1immunoprecipitated with antibody against SSeCKS showedthe same interaction between them (Fig. 5a and b). Quan-titative densitometric analysis from three replicates demon-strated that the amount of co-immunoprecipitation betweenSSeCKS and cyclin D1 after TNF-a treatment increasedsignificantly relative to the control group (Fig. 5c). Next, thelocalization of cyclin D1 in SCs was investigated. UponTNF-a treatment, a significant decrease of cyclin D1 in thenucleus was observed in non-transfected cells (Fig. 6a and

(a) (b)

(d)(c)

Fig. 4 SSeCKS down-regulated cyclin D1 expression in TNF-a trea-

ted SCs. (a) SCs were cultured in concentrations TNF-a for 12 h, and

whole cell lysates were prepared. After adjusting the total protein

concentration, 10 lg proteins were analyzed for cyclin D1. GAPDH

was re-blotted on each lane as a loading control. Blot for cyclin D1

was quantified by densitometry and expression levels relative to

GAPDH were calculated as a percentage of untreated control. (b) SCs

were treated with 10 lM of ERK1/2 inhibitor U0126 for 12 h, and

whole cell lysates were blotted with cyclin D1, phosphorylated-ERK1/2

(p-ERK1/2) and total ERK1/2 antibodies. Control cells were incubated

with DMSO vehicle for the same period of time. (c) Western blot

analysis examined the expression of cyclin D1 and ERK1/2 activities

in non-specific siRNA and SSeCKS knock-down SCs with or without

TNF-a (10 ng/mL) treatment. (d) Cyclin D1 promoter vector and non-

specific siRNA or SSeCKS siRNA vector con-transfected SCs were

cultured in the absence or presence of TNF-a (10 ng/mL) for 12 h.

Lysates were used to assay luciferase activity. Dual luciferase assay

data were reported as ratios of firefly/Renilla luciferase activity in

each sample. In SSeCKS-knockdown SCs, luciferase activities were

significantly reversed. Values were averaged from duplicate experi-

ments. Statistical differences compared with the untreated cells

were given as *p < 0.05 and compared with the TNF-a-treated non-

transfected SCs were given as #p < 0.05.



(a) (b)

(c)

Fig. 5 The co-immunoprecipitation between cyclin D1 and SSeCKS

in SCs. SCs were cultured in the medium with or without 10 ng/mL

TNF for 12 h. (a) cyclin D1 copurifies with SSeCKS in SCs. Samples

were subjected to immunoprecipitation using antibody against

SSeCKS. (b) SSeCKS copurifies with cyclin D1 in SCs. The lysates

were immunoprecipitated with antibodies against cyclin D1. ‘Input1/2’

represents 10% of total protein which got from untreated cells or

TNF-a-treated SCs that used in immunoprecipitation. (c) Quantitative

density analysis of interactions between cyclin D1 and SSeCKS.

Bands were scanned and intensities were expressed as mean ±

SEM (x-fold to normal) for three independent experiments. * indi-

cates that the co-immunoprecipitation of both cyclin D1 with SSeCKS

and SSeCKS with cyclin D1 increased significantly in the TNF-a-

treated SCs. *p < 0.05.

(a) (a)

(b) (d) (f)

(c) (e)

(c)

(b)

Fig. 6 SSeCKS prevents nuclear localization of cyclin D1 in TNF-a

treated SCs. (a) Equal amounts of cytosolic and nuclear lysates from

SCs treated or untreated with 10 ng/mL TNF-a were analyzed by

western blot to detect cyclin D1 localization. Lamin B or a-tubulin were

used as an internal control for nuclear or cytosolic protein, respec-

tively. Blot for cyclin D1 was quantified by densitometry and expres-

sion level relative to Lamin B or a-tubulin were calculated as

percentages of untreated SCs. (b) SCs were cultured in concentra-

tions TNF-a for 12 h, and Cyclin D1 (green) and SSeCKS (red)

localizations were analyzed by fluorescein isothiocyanate (FITC)/tet-

raethyl rhodamine isothiocyanate (TRITC)-labeled antibodies. (i, ii)

normal SCs. (iii, iv) TNF-a treated SCs. (v, vi) SSeCKS siRNA SCs

treated by TNF-a. (c) Non-specific siRNA and SSeCKS knock-down

SCs were cultured in the medium with or without TNF-a (10 ng/mL) for

12 h. Equal cytosolic and nuclear lysates were analyzed with cyclin D1

antibody. Lamin B or a-tubulin was used as a control for nuclear or

cytosolic protein, respectively. Immunoblots were quantified and

average pixel intensity was measured for each band. The chart bar

showed the ratio at each concentration points. The data were

means ± SEM for three independent experiments. Nuclear localized

cyclin D1 was increased about 2-folds in SSeCKS-knockdown cells.

*p < 0.01. Scale bar: 20 lm.


652 | T. Tao et al.

b). However the western blot and immunofluorescenceanalysis revealed that nuclear-localized cyclin D1 increasedto about 2-folds in SSeCKS-knockdown SCs as compared tonormal cells after exposure to 10 ng/mL TNF-a treatment,.But the cyclin D1 localization had no differences between thenon-specific siRNA transfected and un-transfected SCs in theabsence of TNF-a (Fig. 6b and c).

Discussion

The present study demonstrated that TNF-a potentiallyinhibited SC proliferation, up-regulated SSeCKS expressionand down-regulated cyclin D1 expression in a dose-depen-dent manner. In the circumstance in which SSeCKS wasknockdown, SCs became less sensitive to TNF-a duringgrowth arrest as compared with control cells. Furthermore,we revealed that knockdown of SSeCKS expression canprompt cyclin D1 redistribution to the nucleus and up-regulate its expression in TNF-a-treated SCs.

The pro-inflammatory cytokine, TNF-a, has been describedas one of the central injury-induced cytokines responsible fora series of disease defining events during Wallerian degen-eration (Hartung et al. 1992). TNF-a generation and secretionare also central to set the cytokine network through theprocess (Schafers et al. 2003). The molecular signalingcascade leading to the growth arrest of SC in vivo remainselusive (Bonetti et al. 2000; Kury et al. 2003). Similar to apervious study, we report that TNF-a potentiates SC growtharrest in vitro in a dose-dependent manner (Chandross et al.1996). Although our research did not confirm that TNF-apotentiates SC death in vivo as reported by Boyle et al., thedifference was that the later study was performed in serum-deprived cells (Boyle et al. 2005).

Src-suppressed protein kinase C substrate, originallyidentified as a transcriptionally-suppressed gene in v-srcand ras-transformed rodent fibroblast cells (Lin et al. 1995),is the orthologue of the human AKAP12 gene that encodes akinase-scaffolding protein (Nauert et al. 1997). Re-expres-sion of SSeCKS to physiologic levels in ras-transformedfibroblasts or epithelial prostate cancer cells suppressedmorphological transformation, anchorage- and growth factor-independent proliferation, and metastatic potential. It alsorestored normal actin-based cytoskeletal architecture andcell-cycle controls on cyclin D1 expression (Lin and Gelman1997; Xia et al. 2001). Here we provided new evidenceshowing that TNF-a up-regulated the expression of SSeCKSin SCs in a dose-dependent manner that was similar inastrocytes and C6 glioma cells (Sun et al. 2007a; Yan et al.2007). Furthermore, the concentration-dependent increase inSSeCKS mRNA and protein levels were accompanied by areduced expression of cyclin D1.

As cyclin D1 has been reported to interact with SSeCKSboth in vitro and in vivo (Nelson and Gelman 1997; Lin andGelman 2002), we went on to demonstrate the association

of SSeCKS and cyclin D1 in normal and TNF-a-treatedSCs. Cyclin D1 is a regulator of the CDK family of serine/threonine kinases that play a pivotal role in promoting G1to S phase progression (Pines and Hunter 1991; Baldinet al. 1993). In NIH3T3 cells, over-expression SSeCKScould down-regulate cyclin D1 expression, and arrest cellsin G1 phase (Lin et al. 2000). Upon TNF-a treatment, theexpression of cyclin D1 decreased in SCs. FollowingSSeCKS knockdown in SCs, cyclin D1 levels was notablyincreased. Sustained activation of the ERK subfamily isrequired for the induction of cyclin D1 in early G1 phase(Lavoie et al. 1996; Assoian 1997). Previous studies havedemonstrated that ERK1/2 activation was important for SCproliferation (Monje et al. 2006). ERK1/2 activity wassignificantly suppressed in TNF-a treated SCs, and itsactivity was significantly reversed in SSeCKS knockdownSCs. These data indicate that suppression of ERK1/2activity by SSeCKS reduced cyclin D1 expression in TNF-a-treated SCs. Why wasn’t the activity in untreatedSSeCKS knock-down SCs changed compared with thecells with intact SSeCKS levels? Recent work has linkedactivation of the MAPK/ERK kinase (MEK)-ERK1/2pathway with the PKC pathway and TNF-a could activatedPKC in SCs (Scarpini et al. 1999). Thus, it is possible thatalthough SSeCKS’s expression was knocked down in theabsence of TNF-a, PKC activation and consequently cyclinD1 expression remained unchanged in these SCs.

Strong nuclear cyclin D1 staining was restricted to cells inthe G1 phase. Cyclin D1 translocates to the cytoplasm duringS phase (Baldin et al. 1993). Cyclin D1 may help maintain-ing a differentiated state by forming a complex with CDK4and CDK6, thereby preventing cell cycle progression (Lewand Wang 1995). In vivo, most proliferating SCs showednuclear cyclin D1 expression during Wallerian degeneration,although some cells display a lower intensity cytoplasmicstaining while others yet were completely devoid of cyclinD1 (Atanasoski et al. 2001). In our study, we found that lesscyclin D1 localized in the nucleus after TNF-a treatment.SSeCKS was reported to have the capacity to bind to cyclinD1 in quiescent NIH3T3 cells, whereas this binding wasdisrupted in proliferating cells (Lin et al. 2000; Lin andGelman 2002). In the TNF-a-treated SCs, while proliferationhad been suppressed, the binding ability of cyclin D1 withSSeCKS was higher than that in the proliferating SCs, whichwas similar to that of hepatocellular carcinomas cell(Yamamoto et al. 2006). The SSeCKS gene encodes twopotential cyclin-binding motifs (CY) flanking major in vivoPKC phosphorylation sites (Ser507/515). Pre-phosphoryla-tion of SSeCKS by PKC results in a rapid translocation ofcyclin D1 to the nucleus even when over-expressed (Lin andGelman 2002). In TNF-a-treated growth-arrested SCs,phosphorylation of SSeCKS was increased and the phos-phorylation sites (Ser507/515) might be exposed to bindcyclin D1, and anchored it in the perinuclear region. Previous



studies have reveled that decreasing the level of ectopicSSeCKS rescued G1 arrest and the nuclear translocation ofcyclin D1 (Lin et al. 2000). In our studies, knockdownendogenous SSeCKS did not increase SC proliferation andcyclin D1 nuclear translocation. It is possible that inuntreated SSeCKS knockdown SCs, the activity of PKChad no change and the phosphorylation did not increase inthese SCs. Therefore the binding of SSeCKS with cyclin D1was still lower than the quiescent SCs, and knocking downendogenous SSeCKS expression did not increase cyclin D1nuclear localization. So the proliferation had no significantcharge in TNF-a untreated SCs.

In summary, we provide a comprehensive temporal studyof the responses of isolated SCs to TNF-a leading to growtharrest. TNF-a promotes SSeCKS expression, which preventscyclin D1 redistribution to the nucleus and down-regulationof its expression. This synergism between cyclin D1 andSSeCKS in TNF-a-treated SCs may provide a new venue tostudy the proliferation of SCs after nerve injury. Recentstudies have revealed that TNF-a is a direct toxin inducingdemyelination in vivo and in vitro (Lu et al. 2007; Taylor andPollard 2007). Furthermore, allowing axon regrowth wouldstop proliferation and start remyelination. In future studies, itwould be interesting to determine whether SSeCKS partic-ipates in the remyelination caused by injury and whetherSSeCKS is involved in demyelination.

Acknowledgements

The authors wish to thank Dr. Jean-Pierre Montmayeur and Dr.

Yuxiang Hu for helpful criticism and linguistic revision of the

manuscript. This work was supported by the National Natural

Science Foundation of China (No.30300099, No.30770488 and

No.30870320); Natural Science Foundation of Jiangsu province

(No.BK2006547, No.BK2009156 and No.BK2009157); Health

Project of Jiangsu Province (H200632); Special Research Grant

(XK200723) for the Key Laboratory from the Department of Health,

Jiangsu Province; The Society and Technology Grew Project of

Nantong City (S2008020).

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Appendix S1. Supplementary materials and methods.

Figure S1. Brdu labeling assay showed the proliferation of SCs

deprived from forskolin for 1 day or 1 week.

Table S1. Flow cytometry showed that the distribution of cell

cycle have no significantly differences in the normal and 1 day

forskolin deprived SCs, but more cells were arrest in G1 phase when

deprived for 1 week.

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Tumor necrosis factor-alpha inhibits Schwann cell proliferation by up-regulating Src-suppressed protein kinase C substrate expression