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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2001 as doi:10.1096/fj.01-0434fje. |
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Department of Bioengineering and The Whitaker Institute of Biomedical Engineering, University of California, San Diego, La Jolla, California 92093, USA
3Correspondence: Department of Bioengineering and The Whitaker Institute of Biomedical Engineering, University of California, San Diego, 9500 Gilman Dr., MC 0427, La Jolla, CA 92093, USA. E-mail: shuchien{at}ucsd.edu
SPECIFIC AIMS
We tested the hypothesis that the endothelial cell (EC) membrane lipid bilayer can transduce mechanical stimuli into intracellular signaling. The testing of this hypothesis led to two specific aims: 1) to determine the effects of different temporal modes of shear stress on the dynamics of membrane lipids and on the kinase activities of two signaling molecules in the mitogen-activated protein kinase (MAPK) family, i.e., extracellular signal-regulated kinase (ERK) and c-jun amino-terminal kinase (JNK); and 2) to assess the effects of a membrane fluidizer (benzyl alcohol) and an agent that reduces fluidity (cholesterol) on the MAPK activities.
PRINCIPAL FINDINGS
1. The effects of shear stress (
) on cell membrane fluidity are time dependent and spatially heterogeneous
We first subjected confluent ECs to a step- of 10 dynes/cm2 and measured the time course of changes in the normalized diffusion coefficient, D*, of the fluorescent lipoid molecule, DiI, in the apical cell membrane upstream to and downstream from the nucleus [D* = (D/Dinit)exp-(D/Dinit)control, where ‘exp’ refers to the effects of shear stress and ‘control’ refers to nonsheared control samples; see Fig. 1
A]. D was measured using fluorescence recovery after photobleaching on a confocal laser-scanning microscope. D is used as an index of fluidity and to represent the DiI diffusion coefficient in the outer leaflet of the apical plasma membrane. Figure 1A
shows the effects of step- to 10 dynes/cm2 on the time course of D*. On the upstream side of the cell, D* increased significantly over initial values within 5 s (D*=0.39±0.13) and returned to initial values by 10 s. On the downstream side, D* decreased within 5 s (D=*-0.22±0.16), remained low for 30 s, and returned to initial values by 1 min (n=7). These results are consistent with our earlier reports on the time- and position-dependent effects of step- on DiI diffusion (Butler et al., 2001, Am. J. Physiol. 280, C962–C969). Differences between up- and downstream values of D* were significant at the 5, 10, and 30 s time points.
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2. The endothelial cell lipid bilayer is sensitive to temporal shear gradients
In contrast to the effects of step-, when was linearly ramped to 10 dynes/cm2 at a rate of 20 dynes/cm2 per min, D* decreased significantly by 5 s (D*=-0.27±0.06 and -0.20±0.11, for up- and downstream measurements, respectively) and remained below initial values thereafter (Fig. 1B
). There were no significant differences between the effects of ramp- on upstream and downstream D* values. When the same ramp rate of 20 dynes/cm2 per min was used to increase from 0 to 20 dynes/cm2, the changes in D* were similar to those for a ramp to 10 dynes/cm2 (data not shown).
3. MAPKs are differentially activated by temporal shear gradients
We investigated the rate sensitivity to of ERK and JNK kinase activities in BAECs. Densitometric values of kinase assays are normalized to nonsheared controls. Figure 2
shows that step- elicits a significant increase in the kinase activities of both ERK (1.83±0.08-fold, Fig. 2A
) and JNK (1.83±0.16-fold, Fig. 2B
) and that ramping to the same maximum abrogated this response (n=4, P<0.01).
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4. Benzyl alcohol increases and cholesterol reduces MAPK activity
To assess whether the alterations in membrane fluidity observed after shearing would lead to MAPK activation, we treated confluent BAECs with 45 mM benzyl alcohol (BA), which is a known membrane fluidizer, and 0.1 mM cholesterol, which is known to reduce membrane fluidity. BA treatment induced an increase in ERK activity by 5 min (2.41±0.45-fold), and the value returned to control level by 30 min. JNK activity remained at the control level at 30 min and became significantly elevated (2.90±0.47-fold) by 2 h (n=4, P<0.01). Cholesterol treatment of BAECs for 3 h resulted in significant reductions in the kinase activities of both ERK (0.70±0.06-fold) and JNK (0.71±0.10-fold) (n=3, P<0.05).
CONCLUSIONS
It is appreciated that the ECs, which form the lining of the vasculature, use the temporal aspects of these hemodynamic forces as a signal to maintain vascular homeostasis and vascular tone and that derangements of EC signaling mechanisms may play a role in atherogenesis and the pathologies related to reperfusion of ischemic tissue. Since the EC membrane is a repository for many mechanosensitive molecules and is in direct contact with the circulating blood, it is a likely candidate as a mechanotransducer. Furthermore, membrane fluidity, which is a microrheological property of the lipid bilayer, is known to have a significant modulating effect on membrane protein functions.
The results of the present investigation into rate sensitivity of the membrane lipid bilayer and into intracellular signaling support the hypothesis that direct perturbation of the apical cell membrane, as reflected by changes in membrane fluidity, is a proximal signal transduction event (Fig. 3
). The response of the membrane to fluid forces exhibits the salient features of sensitivity to flow direction, rate of change and magnitude, transience of the response, and the ability to transduce outside shear stimuli to intracellular signals.
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A step- caused an increase in D* on the upstream side of the cell and a concomitant decrease on the downstream side, with the difference lasting for 30 s, suggesting the sensitivity of ECs to flow direction and their ability to buffer the effects of sudden changes in fluidity. This fluidity gradient may be a cue to the cell to sense the direction of flow. The absence of a fluidity gradient across the cell as well as MAPK activation when was ramped provides further evidence that the fluidity gradient may play a role in activation of EC signaling.
The changes in D* due to step- were potent and transient, whereas changes in D* due to ramp- were modest and sustained. These results suggest that the cells as a system are sensitive to rapid changes in and that the membrane lipid bilayer in particular can discriminate between different temporal modes of shearing. Such differential effects on lipid dynamics by differing modes of could result in the activation (or deactivation) of membrane-bound proteins.
Membrane fluidity and ERK and JNK activities are increased by step- but not by ramp-. These results suggest that intracellular signaling in ECs is sensitive to the temporal shear gradient. Benzyl alcohol, a membrane fluidizer, increases ERK and JNK activities. Cholesterol, a potent reducer of fluidity, decreases both ERK and JNK activities. Taken together, these results suggest that rate sensitivity is linked to membrane fluidity and that perturbations in membrane fluidity lead to changes in MAPK activities.
Though the mechanism of the fluidity-induced protein activation is still not known, the present research suggests that the -induced membrane perturbation precedes the mechanotransduction events that involve membrane proteins and intracellular signaling proteins. The alteration of lipid dynamics by may lead to conformational changes in integral membrane proteins (e.g., G-protein-coupled receptors, ion channels, integrins, etc.) or peripheral proteins (G-proteins), and these changes may in turn initiate intracellular signaling (e.g., MAPKs) and gene expression (e.g., MCP-1). The rheological properties of membrane lipids may also modulate the formation of cholesterol-rich microdomains (i.e., rafts, focal adhesions, and caveoli) in which important signaling molecules are clustered.
The present study is the first to demonstrate that the cell membrane is sensitive to the rate of change of shear stress and that shear-induced perturbations of the membrane are sufficient to initiate signal transduction. It is also the first study to show that shear-induced JNK activation is rate sensitive. Thus, this study serves to enhance our understanding of mechanotransduction in the endothelium and the consequent modulation of vascular function.
In conclusion, our results support the hypothesis that -induced changes in lipid dynamics in the plasma membrane is a mechanistic link between mechanical force and chemical signaling. We found that temporal shear gradients are important stimuli for both the position and magnitude dependence of -induced fluidity changes. We also showed that temporal shear gradients determine the magnitude and time course of MAPK activation, which is an important signaling pathway that mediates the -induced modulation of the expression of genes related to vascular function. These results have important implications in understanding the observed correlation between temporal and spatial fluctuations in shear stress and the focal nature of atherosclerotic lesions.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0434fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0434fje 
2 Current address: Department of Bioengineering, The Pennsylvania State University, 205 Hallowell Bldg., University Park, PA 16802, USA. E-mail: pjbbio@engr.psu.edu
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