Mechanical Strain Regulates Osteoblast Proliferation Through Ca2+-CaMK-CREB Signal Pathway

Chin Med Sci J June 2016

Vol. 31, No. 2 P. 100-106

CHINESE MEDICAL SCIENCES JOURNAL ORIGINAL ARTICLE

Mechanical Strain Regulates Osteoblast Proliferation Through Ca2+-CaMK-CREB Signal Pathway△ Yong Guo1, 2, Qi Lv3, Xian-qiong Zou1, Zhi-xiong Yan1, and Yu-xian Yan1, 3* 1

Depantment of Bioengineering, College of Biotechnology, Guilin Medical University, Guilin, Guangxi 541004, China 2

Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China 3

Experiment Management Center, Logistical College of People Armed Police Forces, Tianjin 300162, China

Key words: mechanical strain; calcium; phospholipase C; proliferation; cAMP response element binding protein Objective To investigate the effects of mechanical strain on Ca2+-calmodulin dependent kinase (CaMK)-cAMP response element binding protein (CREB) signal pathway and proliferation of osteoblasts. Methods Using a four-point bending device, MC3T3-E1 cells were exposed to mechanical tensile strains of 2500 μs and 5000 μs at 0.5 Hz respectively. The intracellular free Ca2+ ([Ca2+]i) concentration and calmodulin activity were assayed by fluorospectrophotometry, CaMK II β, CREB, and phosphorylated (activated) CREB (p-CREB) were assessed by Western blot, and cells proliferation was assayed with MTT. Pretreatment with verapamil was carried out to block Ca2+ channel, and inhibitor U73122 was used to inhibit phospholipase C (PLC). Results Mechanical strains of 2500 μs and 5000 μs for 1 to 10 minutes both increased [Ca2+]i level of the cells. The 2500 μs strain, a periodicity of 1 h/d for 3 days, activated calmodulin, elevated protein levels of CaMK II β and p-CREB, and promoted cells proliferation, which were attenuated by pretreatment of verapamil or U73122. The effects of 5000 μs strain on calmodulin, CaMK II β, p-CREB and proliferation were contrary to 2500 μs strain. Conclusion The mechanical strain regulates osteoblasts proliferation through Ca2+-CaMK-CREB signal pathway via Ca2+ channel and PLC/IP3 transduction cascades.

Chin Med Sci J 2016; 31(2):100-106 Received for publication July 06, 2015. *Corresponding author Tel: 86-773-3680651, Email: [email protected] △Supported by the National Natural Science Foundation of China (11432016, 31370942, 11372351), and Higher School Science Foundation of Guangxi (04020150032).

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101

ECHANICAL stimulus plays an important role

CREB and phosphorylated-CREB (p-CREB), and proliferation

in regulating bone growth and adaptation,

of osteoblasts.

which promotes bone formation and suppresses bone resorption.1, 2 Mechanical forces

have been shown to activate many types of signal

MATERIALS AND METHODS

transduction cascades in bone tissue, from increases in

Cell culture

intracellular adenosine triphosphate (ATP), calcium and

A mouse pre-osteoblastic cell line, MC3T3-E1 cell provided

guanine regulatory proteins to activating mitogenactivated

by the School of Basic Medicine of Peking Union Medical

protein kinase (MAPK) and nitric oxide signaling and so on.3, 4

College (Beijing, China), which has been shown to differen-

Osteoblast, the bone-forming and bone-remodeling

tiate into osteoblasts and osteocytes,20, 21 was cultured in

cell, is sensitive to mechanical stimulus and involved in

alpha minimal essential medium (α-MEM, Invitrogen) con-

mechanical signal transduction.5 Previous studies indicated

taining 10% fatal bovine serum and 1% penicillin-strep-

that certain mechanical strain promoted osteoblasts prolife-

tomycin, at 37˚C in humidified 5% CO2 atmosphere and

6-8

Many signaling pathways

95% air. The pharmacological agents used to evaluate the

mediate osteoblasts proliferation, such as ERK1/2 signaling,

potential role of signaling pathways in mechanical respo-

ration and metabolic activity.

6, 9-11

estrogen receptor signal, PI3K-Akt signaling and so on. 2+

nse were Ca2+ channel blocker verapamil (Sigma Aldrich,

Calcium ion (Ca ) is an important and prevalent

20 µmol/L) and PLC inhibitor U73122 (Axon Medchem,

second messenger of signal transduction, and an increase

5 µmol/L). They were added to cell culture two hours prior

2+

in intracellular free calcium ([Ca ]i) concentration is the

to mechanical strain application and remained in the

earliest cellular response detected in mechanically stimulated

culture media throughout the experiment.

12

osteoblasts.

Activation of phospholipase C (PLC)-IP3 and

Ca2+ channel leads to an increase of [Ca2+]i.13 The [Ca2+]i

Application of mechanical tensile strain to cells

activates calmodulin (CaM) which further activates Ca2+-

Mechanical tensile strain was generated by a customized

calmodulin dependent kinase (CaMK), then the activated

four-point bending device (provided by the Institute of

CaMK results in phosphorylation (activation) of cAMP response

Medical Equipment, Academy of Military Medical Sciences,

element binding protein (CREB).

Tianjin, China) as described previously.22 MC3T3-E1 cells

14

Fluid shear stress induced phosphorylation of CREB in 15

were seeded at the density of 2.5×104 cells/cm2 in the cell

and cyclic mechanical stetch

culture dishes and cultivated until they reached 80%

(1 Hz and 5% elongation) induced CREB phosphorylation in

confluence. The cells were subjected to mechanical strain

osteoblastic MC3T3-E1 cells

16

In addition, fluid

of 2500 µs or 5000 µs at 0.5 Hz with a periodicity of 1 h/d.

shear stress rapidly increased the intracellular [Ca2+]i

Unstrained (control) cultures were incubated under the

17

concentration and nitric oxide synthesis in osteoblasts,

same conditions for the maximum period of mechanical

and mechanical tensile strain also resulted in influx of

strain application.

all CREB-positive cardiac fibroblasts.

extracellular Ca

2+

2+ 18

and mobilization of intracellular Ca .

Mechanical stretch induced calcium to enter into cytosol

[Ca2+]i content assay

from the extracellular environment, which resulted in

The MC3T3-E1 cells were stimulated with mechanical strain

19

calcium oscillations in human mesenchymal stem cells.

of 2500 µs or 5000 µs at 0.5 Hz for 1, 3, 5, 10 minutes

However, how calcium-CaMK-CREB signal pathway involved

respectively, then the [Ca2+]i content of the cells was

in mechanotransduction of osteoblasts is still poorly

measured on the control and stimulated cells, as described

understood, and it has not been elucidated fully how

above. After washing with PBS, the cells were scraped off

mechanical stimuli affects the signal pathway.

with a cell scraper, centrifugated and incubated at 37°C for

Inasmuch as Ca

2+

is a prevalent second messenger and

30 minutes with the calcium-specific dye, Fluo-3 AM (2

calcium-CaMK-CREB signal pathway is an important role of

ng/ml) (Molecular Probes, Inc. USA), which fluoresces only

signal transductions, we hypothesized that calcium-CaMK-

upon chelation of the dye with free Ca2+. Then the cells

CREB signal pathway involved in mechanotransduction of

were re-suspended in HEPES buffered saline (HBS: 130

osteoblasts, then mechanical strain regulates osteoblasts

mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L glucose, 1.0

proliferation through this signaling pathway. The purpose

mmol/L MgCl2, 1.0 mmol/L CaCl2, 25 mmol/L HEPES, pH

of this study was to verify this hypothesis by investigating

7.4) following washing with HBS. [Ca2+]i levels of the cells

2+

the effect of mechanical tensile strain on [Ca ]i concen-

were detected using the fluorescence spectrophotometer

tration, CaM activity, protein expression levels of CaMK Ⅱ,

(TECAN, Austria) with excitation at 488 nm and emission at

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CHINESE MEDICAL SCIENCES JOURNAL

June 2016

530 nm. Results were expressed as relative to control.

(Invitrogen) was used to assay for living cells. In this assay,

CaM activity assay

oxidoreductases. The relative content of formazan product

CaM is an important signal molecular of Ca2+-CaMK-CREB

was detected using an enzyme-linked immunosorbent

signal pathway. After washing with cold PBS, the cells were

assay reader at 490 nm. Therefore, the absorbance (OD

fixed with cold 70% alcohol, washed with cold PBS free of

value) at 490 nm was regarded as relative number of living

Ca2+ thrice, then treated with 1 mmol/L trifluoperazine and

cells and relative activity of cell proliferation.26

the MTT is reduced to formazan by intracellular NAD(P)H-

irradiated for 20 minutes with ultraviolet radiation at 250-256 nm. After washing with PBS free of Ca2+ again, the

Statistical analysis

cells were scraped off with a cell scraper, centrifugated and

Statistical analyses were performed using SPSS 12.0

re-suspended in PBS free of Ca2+. The levels of CaM activity

software (Chicago, IL, USA). The data was presented as

of the cells were assayed using the fluorescence spectro-

mean±standard deviation, and analyzed using one-way

photometer (TECAN) with excitation at 488 nm and

analysis of variance (ANOVA). A value of P<0.05 was considered

emission at 530 nm. Results were expressed as relative to

statistically significance.

control.

RESULTS

Western blot analysis of CaMK II β, CREB and p-CREB CaMK II is a broad-specificity calmodulin-dependent kinase

Mechanical strain increased [Ca2+]i level of MC3T3-

which serves to integrate Ca2+ signaling within the cell.23, 24

E1 cells

CREB requires phosphorylation to become a biologically

As shown in Figure 1, [Ca2+]i level increased significantly.

active transcriptional activator.25 The proteins extracted

The maximum level of [Ca2+]i was detected at three minutes

from MC3T3-E1 cells with radio immunoprecipitation (RAPI)

after 2500 µs strain initiation. One minute after 5000 µs

lysis medium containing protease inhibitors. For each

strain initiation, the maximum level was detected.

sample, 30 μg of total protein was boiled in a reducing sample buffer and separated by sodium dodecyl sulfate

Mechanical strain activated CaM attenuated by vera-

polyacrylamide gel electrophoresis (SDS-PAGE) and electro-

pamil and U73122

transferred onto nitrocellulose membranes (BioTrace NT,

After MC3T3-E1 cells were stimulated with mechanical

USA). After treatment with 5% skim milk, the membranes

tensile strain of 2500 µs for 3 days, the relative level of CaM

were incubated overnight with the primary antibody

activity was increased. Pretreatments of verapamil and

(antibody of CaMK II β purchased from Anbo Biotech Co.,

U73122 both attenuated the increase. In contrast, 5000 µs

Ltd, 1:400; antibodies of CREB and p-CREB purchased

strain lowered CaM activity level (Fig. 2). The result

from Cell Signal Technology Co., Ltd, 1:500) at 4˚C , then

indicated that mechanical strain of 2500 µs activated CaM

incubated with horseradish peroxidase-conjugated secondary

via Ca2+ channel and PLC/IP3 signal pathway.

antibody (Wuhan Boster Bioengineering Co., Ltd, 1:1000) for 30 minutes at 37˚C. The immunoreactive bands were visualized using chemiluminescence detection reagent (Beijing Tiangen Biochemical Technology Co. Ltd, Beijing, China). Densitometric measurement of the protein bands was performed using Image Quant software (GE Healthcare). The expression of glyceraldehyde3-phosphatedehydrogenase (GAPDH) was used as a loading control and the data were normalized against those of the corresponding GAPDH. Results were expressed as relative to control.

Figure 1. The intracellular free Ca2+ [Ca2+]i level assay of MC3T3-E1 cells (n=6). Mechanical strains of 2500 µs and 5000 µs both

Cell proliferation assay After seeded at a density of 2.5 × 104 cells/cm2, the

increased [Ca2+]i level of the cells in short time (from 1 minute to 10 minutes). Both 3 minutes

MC3T3-E1 cells were divided at random into groups which

2500 µs strain and 1 minute 5000 µs strain caused

were then subjected to mechanical strains of 2500 µs or

[Ca2+]i peak.

5000 µs at 0.5 Hz for three days. MTT [3-(4, 5-dimethy-

*

lthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] sulution

between the indicated groups.

P<0.05,

**

P<0.01 compared with the control or

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CHINESE MEDICAL SCIENCES JOURNAL

103

Figure 2. Calmodulin (CaM) activity assay of MC3T3-E1 cells (n=6). Mechanical strains of 2500 µs elevated CaM activity level, and the elevation was attenuated by pretreatments of verapamil and U73122. Application of

Figure 3. Western blotting analysis of Ca2+-calmodulin-depen-

5000 µs strains caused lower CaM activity level than

dent kinase (CaMK) II β (n=5).

the control group, pretreatments of verapamil and

Mechanical strains of 2500 µs elevated protein level

U73122 resulted in lower CaM level in the group of

of CaMK II, and the elevation was attenuated by

5000 µs strains.

pretreatments of verapamil and U73122. Mechanical

*

P<0.05,

**

P<0.01 compared with the control or

between the indicated groups.

strains of 5000 µs decreased the protein levels. *

P<0.05,

**

P<0.01 compared with the control or

between the indicated groups.

Mechanical strain elevated protein levels of CaMK II β and p-CREB weaked by verapamil and U73122 After application of 2500 µs strain to MC3T3-E1 cells, the protein levels of CaMK II β and activated CREB (p-CREB) were elevated. Verapamil pretreatment weakened the elevation, so did U73122 (Figs. 3, 4). However, application of 5000 µs strain caused decrease of the protein levels (Figs. 3, 4). The result indicated that the mechanical strain of 2500 µs increased protein level of CaMK II β and activated CREB via Ca2+ channel and PLC/IP3 signal pathway. Mechanical strain promoted proliferation attenuated by verapamil and U73122. The proliferation assay showed that 2500 µs strain promoted proliferative activity of MC3T3-E1 cells, and the proliferation rate was reduced by pretreatments of verapamil and U73122, the effect of 5000 µs strain on cells proliferation was contrary to 2500 µs (Fig. 5).

Figure 4. Western blotting analysis of cAMP response element binding protein (CREB) and phosphorylated-CREB

DISCUSSION

(p-CREB) (n=5). In different groups, the protein levels of CERB were

Mechanical stimuli is a potent regulator of bone remo-

nearly the same. Mechanical strains of 2500 µs

deling and maintenance of bone mass, and many signaling

elevated protein levels of p-CERB, and the elevation

pathways involve in bone cells, responding to mechanical

was attenuated by pretreatments of verapamil and

strain.

27, 28

However, the molecular events involved in

mechanical signal transduction in osteoblasts are not fully understood.

U73122. Strain of 5000 µs decreased the protein levels. *

P<0.05,

**

P<0.01 compared with control or between

the indicated groups.

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CHINESE MEDICAL SCIENCES JOURNAL

June 2016

ological modeling or remodeling, or causes microdamage that is accumulated resulting in fracture.34,

36-38

Our result

indicated that overloaded strain (5000 µs) caused [Ca2+]i peak more quickly. Our results showed that 2500 µs strain increased CaM activity, elevated protein levels of CaMK II β and p-CREB of MC3T3-E1 cells, which were attenuated by verapamil and U73122 respectively. The results demonstrated that mechanical strain of 2500 µs activated Ca2+-CaMK-CREB signal pathway, and the activation of Ca2+-CaMK-CREB signal pathway was dependent on PLC-IP3 signal pathway Figure 5. The proliferative activity of MC3T3-E1 cells assayed

and Ca2+ channel. Additionally, 2500 µs strain promoted

with MTT (n=6).

cells proliferation, which was also attenuated by verapamil

Mechanical strains of 2500 µs promoted proliferation,

and U73122. Considering the CREB target genes related to

which was attenuated by verapamil and U73122.

cell proliferation and CREB mediated cells growth and

Mechanical strains of 5000 µs decreased proliferation.

proliferation,39,

*

feration is dependent on activation of Ca2+-CaMK-CREB signal

P<0.05,

**

P<0.01 compared with the control or

between the indicated groups.

40

the mechanical strain promoting proli-

pathway. In this study, although 2500 µs and 5000 µs strains both increased [Ca2+]i in short time (5 minutes),

Ca

2+

is a prevalent second messenger of signal trans-

after application of them for 3 days, they had opposite effects

duction in cells, and application of mechanical strain to cells

on Ca2+-CaMK-CREB signal pathway and cells proliferation.

2+

12

results in an increase in [Ca ]i concentration.

Activation

We will go on study to investigate the mechanism.

of PLC-IP3 and Ca2+ channel leads to an increase of 2+

13

[Ca ]i.

Activation of PLC-IP3 signal pathway mobilises

In conclusion, mechanical strain of 2500 µs activates 2+

Ca -CaMK-CREB

signal

pathway

via

PLC-IP3

signal

intracellular Ca2+ reservoir of endoplasmic reticulum.29

pathway and Ca2+ channel to mobilise intracellular and

Additionally, open of voltage-gated calcium channels leads

extracellular Ca2+, which results in enhancing osteoblasts

to the extracellular Ca

2+

influx into the cell.

30

An increase of

proliferation.

[Ca2+]i results in activation of the Ca2+-CaMK-CREB signal pathway.14 Therefore, we supposed mechanical strain

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