ORIGINAL ARTICLE

Down-regulated expression of vimentin induced by mechanicalstress in fibroblasts derived from patients with ossificationof the posterior longitudinal ligament

Wei Zhang • Peng Wei • Yu Chen • Lili Yang •

Cheng Jiang • Ping Jiang • Deyu Chen

Received: 25 July 2013 / Revised: 23 May 2014 / Accepted: 24 May 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Purpose The aim of this study was to investigate the

potential role of vimentin in the signal transduction path-

ways initiated by mechanical stimulation that contribute to

ossification of the posterior longitudinal ligament of the

spine (OPLL).

Methods We investigated the effects of in vitro cyclic

stretch on cultured spinal ligament cells derived from

OPLL (OPLL cells) and non-OPLL (non-OPLL cells)

patients. The expression levels of the osteoblast-specific

genes encoding osteocalcin (OCN), alkaline phosphatase

(ALP), and type I collagen (COL I) were assessed by semi-

quantitative reverse transcription-polymerase chain reac-

tion. Vimentin protein expression in OPLL cells was

detected by Western blotting. Small interfering RNA

(siRNA) interference targeting vimentin was performed in

OPLL cells induced by mechanical stress, and the

expression levels of OCN, ALP and COL I were assessed.

Results In response to mechanical stretch, the expression

levels of OCN, ALP, and COL I were increased in OPLL

cells, whereas no change was observed in non-OPLL cells.

Furthermore, knockdown of vimentin protein expression

by siRNA resulted in an increase in the mRNA expression

levels of OCN, ALP, and COL I.

Conclusion The down-regulation of vimentin induced by

mechanical stress plays an important role in the

progression of OPLL through the induction of osteogenic

differentiation in OPLL cells.

Keywords Ossification � Posterior longitudinal ligament �Fibroblast � Vimentin � Mechanical

Introduction

Ossification of the posterior longitudinal ligament of the

spine (OPLL) is characterized by progressive ectopic bone

formation in the spinal ligament. OPLL compresses the

spinal cord and its roots, leading to various degrees of

neurological symptoms that range from discomfort to

severe myelopathy. The precise pathogenesis of OPLL

remains obscure, although multiple contributing factors

have been proposed, including genetic factors, dietary

habits, metabolic abnormalities, and some local factors [1–

3]. Clinical evidence supports the hypothesis that

mechanical stress is one of the local factors that plays an

important role in OPLL progression [4–6]. Mechanical

stimulation activates signaling pathways [7] that ultimately

regulate gene expression and protein synthesis; however,

the mechanisms by which mechanical stress elicits its

effects on spinal ligaments are not well understood.

Vimentin is a member of the intermediate filament

protein family, which together with microtubules and actin

microfilaments makes up the dynamic cytoskeleton that

maintains cell shape, enables intracellular transport, and

supports cell division [8–10]. Many studies have demon-

strated that vimentin performs a number of critical func-

tions involved in adhesion, migration, and cell signaling [9,

11, 12]. In addition, the expression of vimentin is associ-

ated with osteoblast differentiation [13–15]. Down-regu-

lation of vimentin by small interfering RNA (siRNA) has

W. Zhang � P. Wei � C. Jiang � P. Jiang

Department of Orthopedics, Affiliated Hospital of North Sichuan

Medical College, North Sichuan Medical College,

Nanchong 637007, China

Y. Chen � L. Yang � D. Chen (&)

Department of Orthopedics, Chang Zheng Hospital, Second

Military Medical University, Shanghai 200003, China

e-mail: tmmuvip@163.com

123

Eur Spine J

DOI 10.1007/s00586-014-3394-8

been shown to induce endogenous osteocalcin (OCN)

transcription in immature osteoblasts. Conversely, ectopic

over expression of vimentin in osteoblasts inhibits osteo-

blast differentiation, as shown by lower alkaline phospha-

tase activity, delayed mineralization, and decreased

expression of osteoblast marker genes such as bone sialo-

protein and osteocalcin.

Thus, we speculated that mechanical signals may stim-

ulate the release of vimentin, which then participates in the

ossification processes of spinal ligaments. To test this

hypothesis, we examined the effect of mechanical stress on

vimentin protein expression and mRNA expression levels

of the osteoblast-specific genes encoding OCN, alkaline

phosphatase (ALP), and type I collagen (COL I) in spinal

ligament cells derived from OPLL patients (OPLL cells).

We also assessed the effect of RNA interference targeting

vimentin on OCN, ALP, and COL I expression in OPLL

cells.

Materials and methods

Cell culture

Spinal ligament tissues were obtained aseptically from 20

OPLL patients and 13 non-OPLL cervical trauma patients

during surgery. The collected ligaments were minced into

*1 mm2 pieces after washing with phosphate-buffered

saline several times and then placed in 60 mm culture

dishes containing Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10 % heat-inactivated fetal

calf serum (FCS). The explants were incubated in a con-

trolled 95 % air/5 % CO2 atmosphere at 37 �C. The cells

migrating from the explants were harvested with 0.1 %

trypsin, replated in 100 mm culture dishes, and maintained

in DMEM supplemented with 10 % FCS for passage. The

experiments were performed in adherence with the Chinese

National Institutes of Health Guidelines and were approved

by the 2nd Military Medical University Ethics Committee.

Stretch apparatus

Cells at the fifth passage were placed on a 3.5 9 4.0 cm2

silicon chamber coated with 0.1 % gelatin (Flexcell Inter-

national, Hillsborough, NC, USA) at a density of 10,000

cells/cm2. After the cultures reached confluence, the cells

were incubated in DMEM supplemented with 1 % fetal

bovine serum for 24 h and then subjected to cyclic

stretching using a four-point bending apparatus (Flexercell

4000 Tension Plus System, Flexcell International, Hills-

borough, NC, USA) at 120 % peak-to-peak at 0.5 Hz in a

humidified atmosphere of 95 % air and 5 % CO2 at 37 �C.

RNA preparation and complementary DNA synthesis

After 24 h, total RNA was extracted simultaneously from

the cell monolayers with TRIzol reagent (Invitrogen,

Carlsbad, CA, USA), according to the manufacturer’s

protocol. After separation, sedimentation, reduction, and

washing, the RNA concentration was determined. One g of

total RNA was reverse transcribed to produce the first

strand of complementary DNA (cDNA) using a RevertAid

first-strand cDNA synthesis kit (Fermentas corporation,

MD, USA). The product was stored at 20 �C until it was

used for amplification by the polymerase chain reaction

(PCR).

PCR analysis

For PCR amplification, oligonucleotide primers specific to

human sequences were designed based on sequences in

GenBank (Table 1).

After an initial 5 min denaturation step at 94 �C, the

samples were subjected to 35 cycles of denaturation for

30 s at 94 �C; annealing for 1 min at 56.7 �C (OCN),

56.7 �C (ALP), 54.9 �C (COL I) or 59.1 �C (GAPDH);

and extension for 1 min. A final 10-min extension step

at 72 �C was performed. The amplified products were

resolved by electrophoresis in 1.0 % agarose gel with

0.5 mg/mL ethidium bromide. The bands were detected

by an UV illumination of the ethidium bromide-stained

gels, and the intensity was quantified by Quantity One

software (Bio-Rad, CA, USA) for each gene. All of the

products were corrected for the GAPDH mRNA levels.

Western blotting

The cell extracts from cells derived from OPLL and non-

OPLL patients were rotated at 4 �C for 1 h before the

insoluble material was removed by centrifugation at

12,0009g for 10 min. After normalizing for equal protein

concentrations, the cell lysates were resuspended in SDS

sample buffer before separation by SDS-PAGE. The pro-

teins were transferred from the gel to a nitrocellulose

Table 1 PCR primers

OCN 5-AGGGCAGCGAGGTAGTGA-3

5-CCTGAAAGCCGATGTGGT-3

ALP 5-GTGGACTATGCTCACAACAA-3

5-GGAGAAATACGTTCGCTAGA-3

COL I 5-CGAAGACATCCCACCAATC-3

5-ATCACGTCATCGCACAACA-3

GAPDH 5-CGCGGGCTCCAGAACATCAT-3

5-CCAGCCCCAGCGTCAAAGGTG-3

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membrane, and the membranes were subjected to blocking

for 1 h at room temperature in 5 % nonfat dried milk in

Tris-buffered saline Tween-20 buffer (Ximei Chem Co

Ltd, Shanghai, China) followed by Western blot analysis

with anti-Vimentin antibody (1:750, Chemicon, Temecula,

CA, USA) at 4 �C overnight. The membranes were then

washed and incubated with horse radish peroxidase-con-

jugated goat anti-rabbit immunoglobulin G (1:1,000) for

2 h at room temperature. Then, bound antibody was

revealed using 3,3-diaminobenzidine as the substrate.

Finally, the membranes were dried and scanned using an

Epson Perfection photo scanner (Epson Corporation, CA,

USA). The protein intensities were quantified using

Quantity One software (Bio-Rad, CA, USA). GAPDH

served as the internal control. The value of vimentin pro-

tein expression was reported as the ratio of vimentin per

GAPDH.

Short interfering RNA transfection

The siRNA duplexes used to interfere with vimentin

expression were synthesized by GenePharma (Shanghai,

China). Nonsense siRNA (5-UUAAGUAGCUUGGCCU

UGATdT-3 and 5-UCAAGGCCAAGCUACUUAATdT-3)

served as the negative control. The cells were transfected

with the siRNA duplexes using siRNA transfection reagent

(Invitrogen, Carlsbad, CA, USA), according to the manu-

facturer’s instructions. After transfection for 72 h, Western

blot analysis was used to determine the level of vimentin

expression, as described above.

Statistical analysis

All data were expressed as mean ± standard error of the

mean. Independent sample t tests were used to determine if

there were significant differences between the cells derived

from OPLL and non-OPLL patients. A paired t test was

used to compare OPLL cells subjected to siRNA trans-

fection and nontransfected cells. P \ 0.05 was considered

significant.

Results

Differential expression of OCN, ALP, and COL I

in OPLL cells in response to mechanical stretch

To determine whether the cells derived from OPLL

patients possessed osteogenic characteristics, the expres-

sion levels of the osteoblast-specific genes OCN, ALP, and

COL I were assessed using semi-quantitative RT-PCR. The

expression levels of OCN, ALP, and COL I were

significantly up-regulated in the OPLL cells induced by

mechanical stress compared with those not subjected to

mechanical loading (Fig. 1).

Fig. 1 RT-PCR analysis of ALP, COL I, OCN mRNA expression in

OPLL cells subjected to mechanical stress, demonstrating a signif-

icant time-dependent up-regulation of expression in response to

mechanical stress (P \ 0.01)

Fig. 2 RT-PCR analysis of ALP, COL I, OCN mRNA expression in

non-OPLL cells subjected to mechanical stress, showing no signif-

icant change in expression in response to mechanical stress

(P [ 0.05)

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Differential expression of OCN, ALP, and COL I

in non-OPLL cells in response to mechanical stretch

Semi-quantitative RT-PCR was also used to examine the

effect of mechanical stretching on fibroblasts derived from

non-OPLL patients. There were no changes in the

expression levels of OCN, ALP, and COL I in the non-

OPLL cells in response to mechanical stretching (Fig. 2).

The effect of mechanical stress on vimentin protein

expression in OPLL cells

The effect of mechanical stress on vimentin protein

expression in OPLL cells was assessed by Western blot-

ting. Vimentin protein expression was significantly down-

regulated in the cells subjected to mechanical stress com-

pared with cells not subjected to mechanical stress (Fig. 3).

Knockdown efficiency of siRNA targeting vimentin

siRNA targeting vimentin was designed and transfected

into the fibroblasts from OPLL patients. Seventy-two hours

after transfection, Western blot analysis revealed that

vimentin protein expression was reduced by almost 70 %

in the transfected cells compared with the non-transfected

cells (P \ 0.01) (Fig. 4).

Influence of siRNA targeting vimentin on OCN, ALP,

and COL I expression

To determine whether vimentin plays an important role in

the signaling pathways involved in the ossification pro-

cesses of spinal ligaments and affects OCN, ALP, and

COL I mRNA expression levels in OPLL cells, RNA

Fig. 3 Vimentin protein expression levels in control OPLL cells and

those subjected to mechanical stress as determined by Western blot

analysis. Vimentin proteins levels were significantly lower in OPLL

cells induced by mechanical stress than those not subjected to

mechanical stress (P \ 0.01)

Fig. 4 Western blot analysis showing the knockdown efficiency of

vimentin in cells transfected with siRNA targeting vimentin com-

pared with untransfected cells (P \ 0.01)

Fig. 5 The mRNA expression levels of the osteoblast-specific genes

ALP, COL I and OCN in CCL cells transfected with siRNA, showing

significant up-regulation of ALP, COL I, OCN in the transfected

OPLL cells compared with untransfected cells (P \ 0.01)

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interference targeting vimentin was performed. Seventy-

two hours after transfection with siRNA, the mRNA

expression levels of OCN, ALP, and COL I were assessed

via RT-PCR. The mRNA expression levels of OCN, ALP,

and COL I were significantly up-regulated cells transfected

with siRNA compared with the levels observed in

untransfected cells (Fig. 5).

Discussion

Over the last several decades, the involvement of multiple

etiologic factors in the development of OPLL has been

suggested, including genetic, systemic, and local factors [1–

3]. OPLL often progresses after posterior decompressive

surgery of the cervical spine, such as laminectomy or lam-

inoplasty, which causes cervical instability. This clinical

observation supports the hypothesis that mechanical stress is

an environmental factor plays an important role in the pro-

gression of OPLL [4–6]. Mechanical stress is known to be a

regulator of bone remodeling that increases the number of

osteoblasts and the expression levels of various osteogenic

marker genes, such as ALP, type I COL, and OCN [16].

Mechanical forces applied to cell surfaces activate a variety

of mechanotransducers, including mechanosensitive ion

channels [17, 18], G-protein-coupled receptors [19], cell–

cell adhesion complexes [20], cytoskeleton [21], and focal

adhesion sites [22]. Stimulation of these structures activates

downstream signaling pathways [7] that regulate gene

expression and protein synthesis and ultimately promote

spinal ligament cell differentiation into osteogenic cells. In

the present study, we demonstrated that the mRNA expres-

sion levels of OCN, ALP, and COL I were significantly

increased by mechanical stretch in OPLL cells, whereas no

change in expression was observed in non-OPLL cells.

These results indicate that mechanical stress participates in

the development and progression of OPLL by changing the

expression levels of various genes. However, cyclic

stretching failed to stimulate an increase in the expression of

these osteogenic markers in non-OPLL cells, suggesting that

mechanical stress can induce the progression of OPLL but

does not initiate its development. The results also allow us to

speculate that the metaplasia of OPLL cells into osteogenic

cells has already occurred in OPLL, consistent with other

studies of OPLL pathogenesis [23, 24].

Vimentin is an intermediate filament protein that toge-

ther with microtubules and actin microfilaments makes up

the dynamic cytoskeleton that plays an important role in

mechanotransduction and maintaining cell shape, enabling

intracellular transport, and supporting cell division [8–10].

It has been shown to function as a potential regulator of cell

growth and differentiation. Studies using COS1 monkey

kidney cells and mouse osteoblastic MC3T3-E1 cells have

provided evidence that down-regulation of vimentin by

siRNA induces endogenous OCN transcription in immature

osteoblasts. Conversely, ectopic over expression of

vimentin in osteoblasts inhibits osteoblast differentiation,

as shown by lower ALP activity, delayed mineralization

and decreased expression of osteoblast marker genes such

as bone sialoprotein and osteocalcin [15]. We hypothesized

that the expression of vimentin in OPLL fibroblasts sub-

jected to mechanical stress should be down-regulated.

Indeed, we detected a significant down-regulation of

vimentin in OPLL fibroblasts induced by mechanical stress

compared with that observed in cells not subjected to

mechanical stress. We performed siRNA interfering tar-

geting vimentin in OPLL fibroblasts, and after 72 h of

transfection with siRNA, the mRNA expression levels of

OCN, ALP, and COL I were significantly up-regulated

compared with those observed in non-transfected cells.

Based on these observations, we propose that the

mechanical stress-induced decrease in vimentin expression

plays an important role in the pathogenesis of OPLL.

Additional studies are required to understand the exact

mechanisms by which vimentin contributes to the pro-

gression of OPLL.

Conflict of interest None.

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