The overall objective of the proposed studies is to address the mechanisms by which thrombin, a serine protease released during clotting initiated by sepsis or vascular injury, regulates the expression of adhesive protein intercellular adhesion molecule-1 (ICAM-1;CD54) in endothelial cells, and how this event promotes sequestration and migration of polymorphonuclear leukocytes (PMN) in the lung and thus contributes to development of lung vascular injury. The basis of ICAM-1 expression involves activation of RelA/p65 subunit of the transcription factor NF-(B. Activation of RelA/p65 requires its release from the inhibitory protein I(B( in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the mechanisms of its release have been elucidated, the cytoplasmic events regulating the translocation of RelAp65 to the nucleus remain elusive. We previously showed that activation of RhoA/ROCK and the dynamic changes in actin cytoskeleton induced by thrombin are crucial for NF-(B activation and ICAM-1 expression. We now have evidence that cofilin, an actin binding protein that promotes actin depolymerization, occupies a central position in RhoA-actin pathway mediating ICAM-1 expression by virtue of facilitating the nuclear translocation of RelA/p65. Interestingly, LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot (SSH1L), a cofilin phosphatase, also regulate ICAM-1 expression. Additionally, MLCK and its target myosin IIA play an important role in thrombin-induced NF-(B activation. Based upon these findings, we hypothesize that thrombin engages LIMK1 and SSH1L as well as MLCK to regulate actin-myosin interaction, which in turn facilitates nuclear translocation of RelA/p65, and expression of ICAM-1 in endothelial cells. We will also test the hypothesis that MLCK signaling of ICAM-1-dependent endothelial adhesivity by this mechanism contributes to lung PMN sequestration and PMN-mediated lung vascular injury and tissue edema in mice. We will pursue the following specific aims to test this hypothesis.
Specific Aim 1 will determine the role of LIMK1 and SSH1L in regulating the changes in the actin cytoskeleton leading to nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.
Specific Aim 2 will address the role of MLCK in regulating actin-myosin interaction leading to nuclear transport of RelA/p65 and expression of ICAM-1 in endothelial cells.
Specific Aim 3 will evaluate the in vivo role of endothelial MLCK in regulating thrombin-induced ICAM-1 expression, lung PMN infiltration, and PMN-mediated lung vascular injury in mice. We will use multidisciplinary approaches ranging from biochemical, cellular, and molecular biology to lung physiology to carry out these studies. With the information gained, we believe that it will be possible to block PMN-mediated lung vascular injury by inhibiting the specific signaling events controlling ICAM-1 expression associated with intravascular coagulation and consequent inflammation.
These studies will provide novel insights into the mechanisms regulating translocation of RelA/p65 into the nucleus and thereby expression of ICAM-1, and its consequences on lung PMN sequestration and lung vascular injury. The information gained may lead to the development of strategies for interfering with specific signaling events controlling ICAM-1 expression and thereby preventing or limiting lung PMN uptake and lung vascular injury associated with inflammatory disease states as Acute Respiratory Distress Syndrome (ARDS).
|Bijli, Kaiser M; Fazal, Fabeha; Slavin, Spencer A et al. (2016) Phospholipase C-? signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury. Am J Physiol Lung Cell Mol Physiol 311:L517-24|
|Leonard, Antony; Paton, Adrienne W; El-Quadi, Monaliza et al. (2014) Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation. PLoS One 9:e110949|
|Bijli, Kaiser M; Kanter, Bryce G; Minhajuddin, Mohammad et al. (2014) Regulation of endothelial cell inflammation and lung polymorphonuclear lymphocyte infiltration by transglutaminase 2. Shock 42:562-9|
|Fazal, Fabeha; Bijli, Kaiser M; Murrill, Matthew et al. (2013) Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration. PLoS One 8:e59965|
|Leonard, Antony; Marando, Catherine; Rahman, Arshad et al. (2013) Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-*B activation and endothelial cell inflammation. Am J Physiol Lung Cell Mol Physiol 305:L651-64|
|Bijli, Kaiser M; Fazal, Fabeha; Rahman, Arshad (2012) Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2. Am J Respir Cell Mol Biol 47:660-8|
|Rahman, Arshad; Fazal, Fabeha (2011) Blocking NF-?B: an inflammatory issue. Proc Am Thorac Soc 8:497-503|
|Yang, Liquan; Chen, Guangchun; Mohanty, Sonali et al. (2011) GPR56 Regulates VEGF production and angiogenesis during melanoma progression. Cancer Res 71:5558-68|