Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.
OBJECTIVE
Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms.


RESULTS
Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated.


CONCLUSIONS
We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.
From the Department of Pathology, Metabolic Diseases Institute, University of Cincinnati, OH.
Vidya
V
Gadang
From the Department of Pathology, Metabolic Diseases Institute, University of Cincinnati, OH.
Eddy
E
Konaniah
From the Department of Pathology, Metabolic Diseases Institute, University of Cincinnati, OH.
David Y
DY
Hui
From the Department of Pathology, Metabolic Diseases Institute, University of Cincinnati, OH. anja.jaeschke@uc.edu.
Anja
A
Jaeschke
D000818:Animals; D002339:Carotid Arteries / Q000201:enzymology / Q000473:pathology; D020212:Carotid Artery Injuries / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D006965:Hyperplasia; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000172:deficiency* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D058426:Neointima; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D047428:Protein Kinase Inhibitors; D064067:Rho Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C577427:GEFT protein, mouse; C526380:RhoA protein, mouse
mitogen-activated protein kinase kinase kinase; neointima