HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/8/899 02-0901fjev1    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 AGARWAL, S. Articles by GASSNER, A. S. R. Search for Related Content PubMed PubMed Citation Articles by AGARWAL, S. Articles by GASSNER, A. S. R.

A central role for the nuclear factor-B pathway in anti-inflammatory and proinflammatory actions of mechanical strain ¹

McGowen Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

²Correspondence: McGowen Institute of Regenerative, 589 Salk Hall, University of Pittsburgh, 3501 Terrace St., Pittsburgh, PA 15261-1964, USA. E-mail:sagar{at}pitt.edu

SPECIFIC AIMS

Mechanical signals play an integral role in bone homeostasis. The present report shows that signals generated by tensile strain of low magnitude (TENS-L) are anti-inflammatory and inhibit proinflammatory gene induction, whereas signals generated by tensile strain of high magnitude (TENS-H) induce proinflammatory genes in osteoblast-like periodontal ligament (PDL) cells. The objective of this study was to investigate the mechanisms of intracellular actions of TENS-L and TENS-H. We show that TENS-L and TENS-H exploit the nuclear factor B (NF-B) signal transduction pathway disparately as a central mechanism to modulate proinflammatory gene induction.

PRINCIPAL FINDINGS

1. Tensile strain exerts anti-inflammatory and proinflammatory effects in a magnitude-dependent manner
Tensile forces regulate responses of bone-like PDL cells to interleukin 1ß (IL-1ß), a cytokine known to mediate activation of multiple proinflammatory genes, including cyclooxygenase-2 (COX-2). To examine how various magnitudes of TENS modulate cellular responses, we used rhIL-1ß-dependent PGE₂ production as a criterion of its actions. As expected, IL-1ß induced a marked up-regulation of PGE₂ synthesis. However, concomitant exposure of PDL cells to IL-1ß and TENS-L (2–8% equibiaxial strain) significantly (P0.05) down-regulated IL-1ß-induced PGE₂ production dose-dependently: a 39%, 72%, 86%, and 82% reduction was observed with 2%, 4%, 6%, and 8% TENS, respectively (Fig. 1 A). TENS-L alone did not affect PGE₂ production. In contrast, TENS-H (10–18.5% equibiaxial strain) itself was proinflammatory and induced marked up-regulation of PGE₂. In the presence of IL-1ß, TENS-H exhibited additive effects on PGE₂ production.

View larger version (68K): [in this window] [in a new window]   Figure 1. TENS-L inhibits IL-1ß-induced NF-B nuclear translocation. A) Effect of TENS-L on the IL-1ß-dependent nuclear translocation of NF-B. Cells were exposed to TENS-L (6%) in the presence or absence of IL-1ß for 30, 60, 90, or 120 min. The presence of NF-B in the nuclear extracts by EMSA analysis shows a sustained inhibition of IL-1ß-induced nuclear translocation of NF-B by TENS-L. B) Effect of TENS-L on IL-1ß-induced NF-B nuclear translocation by immunofluorescence. Untreated control cells and cells exposed to TENS-L alone exhibit the presence of NF-B in the cytoplasm, IL-1ß-treated cells show a near-complete nuclear translocation of NF-B, and cells exposed to IL-1ß and TENS-L exhibit the presence of the majority of NF-B in the cytoplasm demonstrating TENS-L-mediated inhibition of IL-1ß-dependent nuclear translocation of NF-B. C) Effect of TENS-L on IL-1ß-induced I-Bß degradation. Western blot analysis of cytoplasmic proteins of PDL cells showing a marked reduction of I-B in IL-1ß-treated cells, and inhibition of IL-1ß-induced I-Bß degradation by TENS-L. D) Effect of TENS-L on IL-1ß-induced I-Bß degradation and resynthesis. PDL cells were treated as described in panel A, and the presence of I-Bß was examined by immunofluorescence using rabbit anti-I-Bß immunoglobulins. IL-1ß induced a marked reduction of I-Bß within 30 min and its resynthesis over the next 90 min. Cells treated with IL-1ß and TENS-L reveal an inhibition of IL-1ß-induced I-Bß degradation by TENS-L over a period of 120 min. B, D) White arrows indicate NF-B and I-Bß; red arrows indicate cellular ß-actin stained with FITC labeled phalloidin. Data represent 1 of 3 separate experiments.

2. Target sites of anti-inflammatory and proinflammatory actions of TENS are located upstream of COX-2 mRNA induction
To determine whether TENS acts on PDL cells at the transcriptional level, we measured COX-2 mRNA expression in the presence and absence of TENS-L. Semiquantitative assessment of PCR products revealed that TENS-L (3%, 6%, and 8%) significantly suppressed rhIL-1ß-induced COX-2 mRNA (53%, 86%, and 82%) within initial 4 h, suggesting target sites of TENS-L actions are located upstream of mRNA expression. TENS-L alone failed to induce COX-2 mRNA expression above that of untreated controls.

TENS-H (15%) rapidly up-regulated COX-2 mRNA expression and PGE₂ synthesis. This indicated that TENS-H acts as a proinflammatory signal and its intracellular target sites are also located upstream of COX-2 mRNA induction.

3. NF-B signal transduction pathway is central to the pro- and anti-inflammatory effects of tensile strain
NF-B is the major transcription factor involved in signal transduction of proinflammatory molecules. The involvement of NF-B as a possible intracellular target of TENS-L attenuation of IL-1ß-induced proinflammatory responses by electrophoretic mobility shift assay (EMSA) revealed a rapid nuclear translocation of NF-B in IL-1ß-treated cells. This IL-1ß-induced NF-B nuclear translocation was markedly inhibited after exposure of cells to TENS-L (6% strain, Fig. 1A ). Subunit structure analysis of NF-B revealed that TENS-L directly inhibits the nuclear translocation of p65 and p50 subunits activated by IL-1ß. Similar to untreated controls, transactivation of NF-B was not observed in cells treated with TENS-L alone. Immunofluorescence confirmed the findings that TENS-L inhibits IL-1ß-induced NF-B nuclear translocation by sequestrating it in the cytoplasmic compartment of cells (Fig. 1B ). Upstream events in NF-B pathway by Western blot analysis revealed that TENS-L alone did not induce I-B degradation. However, TENS-L markedly abrogated IL-1ß-induced I-Bß degradation within the first 30 min (Fig. 1C ). These experiments demonstrate that TENS-L exerts anti-inflammatory effects by inhibiting I-Bß degradation and thus suppressing nuclear translocation of NF-B (Fig. 1D ).

Next we investigated whether the TENS-H-induced expression of COX-2 mRNA is mediated by NF-B transcription factors. EMSA analysis revealed that signals generated by TENS-H (15% strain) were sufficient to induce rapid nuclear translocation of NF-B within 30 min, and the effect was sustained for 180 min (Fig. 2 A, B). In the presence of submaximal concentrations of IL-1ß, the effects of TENS-H were additive. Similar to IL-1ß, TENS-H also induced nuclear translocation of NF-B heterodimers comprised of p65 and p50 subunits. A significant inhibition of the NF-B nuclear translocation and subsequent COX-2 mRNA expression (Fig. 2C ) by caffeic acid phenyl ethylester (CAPE) further confirmed that actions of TENS-H are mediated via NF-B. Collectively these results suggest that like IL-1ß proinflammatory signals generated by TENS-H are also transmitted by nuclear translocation of NF-B.

View larger version (49K): [in this window] [in a new window]   Figure 2. TENS-H induces nuclear translocation of NF-B. A) EMSA analysis showing an increase in the nuclear translocation of NF-B in the presence of 15% TENS or IL-1ß (0.4 ng/mL) and their additive effects by coexposure of cells with TENS-H and IL-1ß. Inset: Supershift EMSA analysis showing p65 and p50 subunits of NF-B are activated by both TENS and IL-1ß. B) Nuclear translocation of NF-B by TENS-H (15%) in the absence or presence of IL-1ß. Cells subjected to treatments described above were incubated with rabbit anti-NF-B immunoglobulins and TRITC-conjugated goat anti-rabbit IgG. Untreated control cells exhibit cytoplasmic NF-B, whereas cells treated with TENS-H induced nuclear translocation of NF-B in the presence or absence of IL-1ß. C) Inhibition of COX-2 mRNA expression by CAPE, an inhibitor of NF-B nuclear translocation, implicating a role for NF-B transcription factors in TENS-H actions.

CONCLUSION

NF-B has an established role in proinflammatory cytokine signaling through its nuclear translocation and subsequent activation of a plethora of proinflammatory genes. We demonstrate that the NF-B signal transduction pathway is central to both proinflammatory and anti-inflammatory signaling of TENS, and emphasize three main points (Fig. 3 ): 1) TENS exerts its effects in a magnitude dependent manner and exploits NF-B transcription factors to mediate its diverse effects; 2) Signals generated by TENS-L abrogate transcriptional activation of IL-1ß-induced proinflammatory genes by abrogating nuclear translocation of NF-B via suppression of I-Bß degradation; and 3) TENS-H induces transcriptional activation of proinflammatory genes by augmenting nuclear translocation of NF-B via I-Bß degradation. TENS-L does not abrogate IL-1 actions by down-regulating IL-1ß receptors. Thus, it is likely that a molecule(s) involved in steps leading from IL-1 receptor complex activation to I-Bß degradation may serve as the target(s) of TENS-L actions in the NF-B signal transduction cascade.

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram describing a central role for NF-B transcription factors in the regulation of anti-inflammatory and proinflammatory actions of TENS. Based on our findings, this overview shows that TENS-L induced anti-inflammatory signals down-regulate proinflammatory gene induction by abrogating IL-1ß-induced I-B degradation and thus NF-B nuclear translocation. On the contrary, TENS-H stimulates I-Bß degradation and thus nuclear translocation of NF-B to up-regulate proinflammatory gene induction.

Considering that mechanical signals play an important role in bone formation, these observations reveal an important mechanism of action of tensile strain that may provide a rationale to explain bone resorption under a field experiencing high magnitudes of strain and bone deposition in fields exposed to physiologic or low magnitudes of strain.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0901fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0901fje

This article has been cited by other articles:

				U. Uhlig, H. Fehrenbach, R. A. Lachmann, T. Goldmann, B. Lachmann, E. Vollmer, and S. Uhlig Phosphoinositide 3-OH Kinase Inhibition Prevents Ventilation-induced Lung Cell Activation Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 201 - 208. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/8/899 02-0901fjev1    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 AGARWAL, S. Articles by GASSNER, A. S. R. Search for Related Content PubMed PubMed Citation Articles by AGARWAL, S. Articles by GASSNER, A. S. R.