Uni-axial Cyclic Stretch Induces Cbfa1 Expression in Spinal Ligament Cells Derived from Patients with Ossification of the Posterior Longitudinal Ligament

Iwasaki, Koei; Furukawa, Ken‐Ichi; Tanno, M.; Kusumi, Tomomi; Ueyama, Kazumasa; Tanaka, Masanori; Kudo, Hiroto; Toh, Satoshi; Harata, Seiko; Motomura, Shigeru

doi:10.1007/s00223-002-0021-1

Cited by 58 publications

(35 citation statements)

References 46 publications

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“…Fibroblasts cells can differentiate into osteoblasts or osteocytes [4,8,9,12,24,28]. This differentiation should be accompanied by an upregulation of several marker genes related to bone formation, such as OCN, ALP and COL I.…”

Section: Discussionmentioning

confidence: 99%

“…Attempts have been made to simulate the mechanical stress in vitro, mostly with a motor-driven uniaxial sinusoidal cyclic stretch [18]. The system induced the transcription of messenger ribonucleic acids of several growth factors, including BMP-2, BMP-4, prostaglandin I 2 synthase, and osteoblast-specific transcription factor in the cells from OPLL patients [9,19,24,30]. At the same time, several studies have suggested that ER stress response can be activated by mechanical stress in some cellular pathological processes, such as apoptosis in rat annulus fibrosus cells or human connective tissue cells [32].…”

Section: Discussionmentioning

confidence: 99%

“…These two processes might differ in terms of the speed of bone formation [6,31]. Although several studies have investigated the osteogenetic characteristic of the spinal ligament fibroblasts from OPLL patients, the intracellular signaling pathways still remain unclear [4,8,9,12,24,28].…”

Section: Introductionmentioning

confidence: 99%

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Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

et al. 2013

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Purpose Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients. Methods Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72 h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group. Results Spinal ligament fibroblasts were observed 7-10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72 h after RNA interference targeting PERK was performed on the cells from OPLL patients. Conclusions The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERKmediated ER stress might be involved in development of OPLL.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

et al. 2013

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“…Furthermore, in patients that underwent anterior interbody fusion, the development of ossification evidently slowed and even stopped [17]. Based on these reports, OPLL may tend to progress at the area where mechanical stretch exists, which has been reported by several authors [5,6,18].…”

Section: Discussionmentioning

confidence: 73%

“…OPLL is a chronic and progressive disease, and clinical symptoms may only occur after the ossified ligaments develop to a certain degree. Although some studies have investigated the role of mechanical strain in OPLL development, the signaling mechanism of such a role remains unclear [5,6].…”

Section: Introductionmentioning

confidence: 99%

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament

et al. 2011

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Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that mechanical strain can lead to the development of OPLL, although the signaling mechanism is not fully understood. Connexin43 (Cx43), a gap-junction protein, has been shown to be of particular importance in bone formation. We hypothesized that Cx43 may play an important role in the signal transmission induced by mechanical strain during the development of OPLL. To explore this possibility, we cultured fibroblasts from spinal ligaments of OPLL and non-OPLL patients and preloaded mechanical stretch onto the cells via a Flexercell 4000 Tension Plus system. We evaluated expression changes in osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 via semiquantitative RT-PCR and western blotting at 12 and 24 h after mechanical strain application in contrast to static conditions. We observed a significant gene up-regulation of OCN, ALP and COL I and Cx43 protein in OPLL cells after mechanical strain application, but no changes in non-OPLL cells. Notably, after RNA interference targeting Cx43 was performed in OPLL cells, we found that there were no significant changes in the expressions of OCN, ALP, COL I and Cx43 after the mechanical strain was applied for 24 h. Thus, we propose that the increase in Cx43 expression induced by mechanical strain in OPLL cells plays an important role in the progression of OPLL.

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Mechanical stress‐mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts

Kanno

Takahashi

Tsujisawa

et al. 2007

J of Cellular Biochemistry

194

159

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The sequence of biochemical events involved in mechanical stress-induced signaling in osteoblastic cells remains unclear. Runx2, a transcription factor involved in the control of osteoblast differentiation, has been identified as a target of mechanical stress-induced signaling in osteoblastic cells. In this study, uniaxial sinusoidal stretching (15% strain, 115% peak-to-peak, at 1/12 Hz) stimulated the differentiation of osteoblast-like MC3T3-E1 cells and rat primary osteoblastic cells by activating Runx2. We examined the involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of Runx2 during mechanical stress. Mechanical stress increased alkaline phosphatase activity, a marker of osteoblast differentiation, increased the expression of the osteoblast-specific extracellular matrix (ECM) protein osteocalcin, and induced Runx2 activation, along with increased osterix expression. Furthermore, activation of ERK1/2 and p38 MAPKs increased significantly. U0126, a selective inhibitor of ERK1/2, completely blocked Runx2 activation during periods of mechanical stress, but the p38 MAPK-selective inhibitor SB203580 did not alter nuclear phosphorylation of Runx2. Small interfering RNA (siRNA) targeting Rous sarcoma kinase (RAS), an upstream regulator of both ERK1/2 and p38 MAPKs, inhibited stretch-induced ERK1/2 activation, but not mechanically induced p38 MAPK activity. Furthermore, mechanically induced Runx2 activation was inhibited by Ras depletion, using siRNA. These findings indicate that mechanical stress regulates Runx2 activation and favors osteoblast differentiation through the activation of MAPK signal transduction pathways and Ras/Raf-dependent ERK1/2 activation, independent of p38 MAPK signaling.

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Uni-axial Cyclic Stretch Induces Cbfa1 Expression in Spinal Ligament Cells Derived from Patients with Ossification of the Posterior Longitudinal Ligament

Cited by 58 publications

References 46 publications

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament

Mechanical stress‐mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts

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