These results show that JNK is activated in a matrix-specific manner in response to fluid shear stress. JNK is activated at early times by acute onset of laminar shear and at later times by oscillatory shear in cells on a FN matrix, but little or no activation occurred in cells on basement membrane protein or type I Coll. JNK activation is also seen in vivo at atheroprone regions of arteries coincident with FN in the subendothelial matrix. Matrix-specific activation of JNK by shear stress requires new integrin ligation, since blocking integrin binding to FN abrogated its activation. JNK activation by shear also requires MKK4 and PAK. Both kinases are activated by acute onset of laminar flow or by oscillatory shear stress in a matrix-specific manner, and inhibiting either kinase blocked JNK activation. Furthermore, a small peptide inhibitor of PAK decreased JNK activation in atheroprone areas of the vasculature in vivo. Taken together, these results suggest that JNK is activated by disturbed flow in ECs adherent to FN in vivo.
JNK is known to play a key role in expression of cytokines and adhesion molecules that mediate leukocyte recruitment and activation 18–21,34
. Inhibiting or deleting JNK also reduced atherosclerosis in ApoE−/−
. JNK can mediate apoptosis under stressful conditions 16
, thus, could contribute to the elevated rates of apoptosis at atheroprone regions in vivo. Taken together, the data therefore suggest that ECM-dependent JNK activity is likely to play a significant role in atherogenesis.
Endothelial cells are normally adhered to a basement membrane in which the major components are LN, which binds integrins α6
, and Coll IV, which binds integrins α2
. Injury, inflammation and angiogenesis that promote vascular remodeling lead to deposition of a provisional matrix containing proteins including FN and FG, which bind primarily α5
. Binding of these integrins to matrix is associated with an activated cell phenotype, with increased cell migration and proliferation 9,38
, consistent with the EC phenotype in atheroprone regions 39,40
. This matrix remodeling most likely contributes to resolving the inflammation or injury in response to acute insults but under chronic stresses may have deleterious aspects.
Previous work in our lab showed that the altered subendothelial matrix at atheroprone sites influences activation of the transcription factor NF-κB, a key regulator of inflammatory gene expression during atherosclerotic progression 7
. We also recently reported that matrix-specific NF-κB activation by flow is mediated by matrix-specific activation of PAK 13
. That JNK, a third key inflammatory mediator, is activated in a matrix- and PAK-dependent manner therefore indicates, first, that matrix remodeling is a major determinant of endothelial activation in this system. It also reveals that the ECM regulates EC phenotype through a network of pathways in which PAK appears to be the central determinant, accounting for both JNK and NF-κB, as well as mediating effects on junctional integrity and permeability more directly.
The result that shear-induced JNK activation is among the events that depend on new integrin ligation also provides additional support for the junctional mechanotransducer model. In this model, integrin activation and ECM binding occur downstream of a junctional complex that stimulates integrin activation through PI3K (). These data fit well with previous results describing a role for PI3K gamma in JNK activation by shear stress 22
. Thus, a series of events beginning with rapid activation of signaling proteins in cell-cell junctions, followed by integrin activation and binding, leading to JNK activation and leukocyte recruitment appear to be components of a single pathway. Our results also show that MKK4 and PAK are upstream of matrix-specific JNK activation. Despite being identified as a potential activator of JNK some years ago 41
, how PAK affects this pathway remains unknown. Upstream kinases such as MEKK1 or scaffold proteins such as JIPs are possible targets. Further work will be required to address this question.
Model for shear-induced JNK activation
Distinct from our results, it has been reported that laminar flow inhibits JNK activation in ECs by inflammatory cytokines such as tumor necrosis factor 42–44
. This effect was proposed to be one of the means by which long term laminar shear is atheroprotective. Under these conditions, inhibition of JNK was mediated through MEK5 and ERK5/BMK1, which inhibited the MAPKKK ASK1 by inducing thioredoxin interacting protein 42,43,45
. These results may appear to conflict with ours and those of other labs that report activation of JNK by flow 20–22,46,47
. However, studies demonstrating suppression of JNK by laminar shear used cells plated on denatured collagen (gelatin). Thus, the matrix specificity of JNK activation may resolve the discrepancy.
In conclusion, our data provide evidence for JNK activation in atheroprone regions of the vasculature through a pathway that involves both disturbed flow and ECM remodeling. Together with published results, these data suggest that JNK contributes to atherogenesis in vivo. These results raise many questions for future work. Which factors determine local PAK activation ECM remodeling in areas of disturbed flow in vivoare major unsolved questions. Although roles for JNK in expression of inflammatory genes such as MCP-1, IL-8, VCAM-1 and prostaglandin D synthase 18,20,48
, and in EC apoptosis and cell migration 15
have been identified, elucidation of the specific roles for JNK during atherogenesis will also be an important area for future research. Understanding the inflammatory pathways controlled by flow may provide novel therapeutic strategies for modulating atherosclerotic progression.