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Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor B in human fibroblast cells¹

HIDEFUMI INOH, NAOKI ISHIGURO, SHIN-ICHI SAWAZAKI, HIDEKI AMMA, MOTOI MIYAZU, HISASHI IWATA, MASAHIRO SOKABE^*, and KEIJI NARUSE^*,2

Department of Orthopedic Surgery,
^* Department of Physiology, Nagoya University Graduate School of Medicine,
Cell Mechano-sensing Project, ICORP, JST, Nagoya 466-8550, Japan

²Correspondence: Department of Physiology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: knaruse{at}med.nagoya-u.ac.jp

SPECIFIC AIMS

The effect of uni-axial cyclic mechanical stretch on the activation of the transcription factor nuclear factor B (NF-B) was investigated in a human fibroblast cell line. We also addressed the involvement of the stretch-activated (SA) channel in stretch-induced NF-B activation.

PRINCIPAL FINDINGS

1. Uni-axial cyclic stretch induces translocation of NF-B into the nucleus
First we examined whether uni-axial cyclic stretch induced translocation of NF-B into the nucleus, because this translocation is known to be an early indispensable event in NF-B activation. Immunostaining of the NF-B subunit RelA showed diffuse staining in the cytoplasm of unstretched cells. In contrast, the cells subjected to uni-axial cyclic stretch for 4 min showed strong staining in the nucleus, indicating that translocation of NF-B into the nucleus was induced by uni-axial cyclic stretch.

To investigate the time course of the translocation of NF-B into the nucleus in response to cyclic stretch, degradation of its inhibitor IB in cytoplasm and accumulation of Rel A in the nucleus were assayed by immunoblot. Figure 1A shows a representative immunoblot stained with anti-Rel A mAb (upper) and anti-actin mAb (lower). Figure 1B shows the time course of NF-B translocation in response to cyclic stretch normalized by actin content. The translocation of NF-B became significant as early as 2 min after the initiation of cyclic stretch, peaked at 4 min, and returned to basal level within 10 min. As IB is dissociated from NF-B and IB degraded in the cytoplasm upon activation of NF-B, we investigated the level of IB in the cytoplasm. Figure 1C shows a representative immunoblot of IB (upper) and actin (lower) in the cytoplasm fraction from the cells subjected to uni-axial cyclic stretch. Figure 1D shows the time course of IB degradation in response to cyclic stretch normalized by actin content. The degradation of IB became significant as early as 1 min after the initiation of cyclic stretch; the IB level reached a minimum at 4 min. These results strongly suggest that cyclic stretch induces translocation of NF-B into the nucleus.

View larger version (26K): [in this window] [in a new window]   Figure 1. The time course of the translocation of NF-B into the nucleus and degradation of IB in the cytosol. A) A representative immunoblot stained with Rel A mAb (upper) and actin mAb (lower). B) The time course of NF-B translocation in response to cyclic stretch normalized by actin content. The translocation of NF-B was detected as soon as 2 min after the initiation of cyclic stretch, peaked at 4 min, and returned to basal level within 10 min. C) A representative immunoblot stained with IB mAb (upper) and actin mAb (lower). D) Time course of IB degradation in response to uni-axial cyclic stretch normalized by actin content. Asterisks denote values significantly different (P<0.05) from control.

2. Involvement of SA channels in the stretch-induced translocation of NF-B
We wanted to investigate the mechanism(s) by which NF-B is activated under cyclic stretch. We used the same protocol as in our previous studies: to examine the effects of the removal of extracellular Ca²⁺ and the application of Gd³⁺, a potent SA channel blocker, on NF-B activation. When the cells were cyclically stretched in the absence of extracellular Ca²⁺ or in the presence of 20 µM Gd³⁺, translocation of NF-B and the degradation of IB were significantly inhibited. Although it is reported that low concentrations of Gd³⁺ do not inhibit Ca²⁺ channels, we further examined the effects of classic Ca²⁺ blockers like nifedipine (10 µM) or verapamil (10 µM), which turned out to have no effect on the translocation of NF-B and degradation of IB. These results suggest that Ca²⁺ influx through the SA channel is critical for translocation of NF-B.

3. Uni-axial cyclic stretch-induced the activation of NF-B, followed by COX-2 mRNA expression
To confirm that NF-B is activated after its translocation into the nucleus, we measured luciferase activity in the cells transfected with pNF-B-Luc. The cells transfected with pNF-B-Luc were cyclically stretched for various periods and incubated for 6 h before the assay. The activity of NF-B as assessed by the luciferase activity became significant 4 min after initiation of cyclic stretch, peaked at 15 min (6.4-fold increase), and decreased gradually at 30 min (Fig. 2 ). These results suggest that cyclic stretch induced NF-B activation after its translocation into the nucleus. The involvement of SA channels was also investigated using the reporter gene assay. In the absence of extracellular Ca²⁺ or in the presence of 20 µM Gd³⁺, cyclic stretch for 15 min did not increase the activity of NF-B (Fig. 2) . The application of IL-1, a positive control, significantly activated NF-B. We examined the effect of NF-B inhibitors such as parthenolide and SN-50 on stretch-induced COX-2 mRNA expression. When the cells were mechanically stretched in the presence of parthenolide (20 µM) or SN-50 (80 µg/ml), stretch-induced expression of COX2 mRNA was significantly inhibited without affecting COX1 mRNA levels.

View larger version (34K): [in this window] [in a new window]   Figure 2. Involvement of SA channels in NF-B activation. In the absence of extracellular Ca2+ or application of 20 µM Gd3+, cyclic stretch for 15 min did not increase NF-B activity. The application of IL-1, which is a positive control, significantly activated NF-B. Asterisks denote values significantly different (P<0.05) from control.

CONCLUSIONS

This study demonstrated that uni-axial cyclic stretch activated the transcription factor NF-B probably via the activation of SA channel in human lung fibroblasts.

NF-B is a powerful transcription factor that can activate the transcription process of various immune and inflammation response genes containing B consensus sequence. We have demonstrated that uni-axial cyclic stretch could activate NF-B. By immunoblotting, translocation of NF-B was first detectable 2 min after the onset of stretch and peaked at 4 min. Luciferase activity in the cells transfected with pNF-B-Luc increased 4 min after the initiation of cyclic stretch and peaked at 15 min. These data indicate that uni-axial cyclic stretch induced translocation and the ensuing activation of NF-B within much shorter period than reported by other groups using different stretching methods.

Activation of nuclear factors by mechanical stimuli to the cells has been reported in several cell types. However, little is known about the mechanism whereby NF-B is activated by mechanical stress.

Ca²⁺ plays a crucial role in stretch-induced cellular responses. Stretch is known to influence ionic fluxes in skeletal muscle cells and to induce secretion of several vasoactive substances probably via Ca²⁺ mobilization in endothelial cells. In human umbilical endothelial cells, uni-axial stretch increased intracellular Ca²⁺ concentration, which was inhibited when the cells were stretched in nominally Ca²⁺-free solution or in the presence of Gd³⁺. It has been suggested that the increase in intracellular Ca²⁺ concentration arises from Ca²⁺ entry through the SA channels in endothelial cells. When cells were stretched in nominally Ca²⁺-free solution or in the presence of Gd³⁺, activation of NF-B was almost completely inhibited. These data strongly suggest that the activation of NF-B is triggered by the intracellular Ca²⁺ increase via SA channels, although we should consider other mechanisms for the stretch-induced Ca²⁺ increase. The exact mechanism of how the elevated level of Ca²⁺ activates NF-B remains unclear. In accordance with our hypothesis, a recent report suggested a relationship between the activation of NF-B and intracellular Ca²⁺. Dolmetsch et al. reported that the amplitude and duration of calcium signals in B lymphocytes control differential activation of the proinflammatory transcriptional regulators NF-B, c-Jun amino-terminal kinase, and NF-AT and that NF-B is selectively activated by a large Ca²⁺ transient. They revealed a mechanism by which a multifunctional second messenger such as Ca²⁺ could achieve specificity in signaling to the nucleus.

Mechanical stretch induces prostaglandin (PG) E₂ production, which is thought to increase in cases of inflammation due to repetitive motion such as tendonitis, bursitis, and fascitis in fibroblast and skeletal muscle cells. We reported earlier that COX-2 expression, which can be induced by inflammatory stimuli, hormones, or mitogens, is up-regulated by uni-axial cyclic stretch probably via the activation of the SA channel in human lung fibroblasts. The COX-2 expression depends on activation of NF-B, because the human COX-2 promoter region contains NF-B. In other words, cyclic mechanical stretch increases the intracellular Ca²⁺ concentration through SA channels and this signal activates NF-B. The activation of NF-B induces expression of COX-2, the first rate-limiting enzyme in the synthesis of PGE₂ in cases of inflammation.

In conclusion, we suggest that uni-axial cyclic stretch induces translocation into the nucleus and activation of NF-B, and that SA channel may be involved in the translocation and activation of NF-B in human lung fibroblasts (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic and hypothetical representation. Uni-axial cyclic stretch induces translocation into the nucleus and activation of NF-B by the increases in [Ca2+]i caused by SA channel activation in human lung fibroblasts.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0354fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0354fje

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This Article Full Text (PDF) All Versions of this Article: 16/3/405 01-0354fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by INOH, H. Articles by NARUSE, K. Search for Related Content PubMed PubMed Citation Articles by INOH, H. Articles by NARUSE, K.