Date Published: March 4, 2014
Publisher: Public Library of Science
Author(s): Michelle Surma, Caitlin Handy, Jiang Chang, Reuben Kapur, Lei Wei, Jianjian Shi, Neil A. Hotchin.
We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1−/− MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
The ROCK family contains two members: ROCK1 and ROCK2, which share 92% identity in the kinase domain –. ROCK plays a critical role in mediating the effects of small GTPase RhoA on stress fiber formation, smooth muscle contraction, cell adhesion, and cell motility , . The best characterized targets of ROCK in the vascular system are myosin light chain (MLC) phosphatase (MYPT), MLC –, and LIM-kinases (LIMK) , . These ROCK-mediated pathways are known to be involved in stress fiber assembly and cell adhesion. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1−/− and ROCK2−/− mice, we recently demonstrated that ROCK1 and ROCK2 can differently regulate stress fiber disassembly and cell adhesion under stress conditions such as cytotoxicity induced by doxorubicin, a chemotherapeutic agent used to treat a wide spectrum of hematologic malignancies and solid tumors for decades, or serum starvation, a frequently used environmental stress in cell biology , . We demonstrated that ROCK2 is required for stabilizing the actin cytoskeleton through regulating cofilin phosphorylation under stress conditions, and in contrast, ROCK1 is involved in destabilizing the actin cytoskeleton through regulating MLC phosphorylation and peripheral actomyosin contraction , . These findings support a novel concept that ROCK1 and ROCK2 can differently regulate stress fiber disassembly, cell adhesion and cell death under stress conditions.
The present study used MEFs derived from ROCK1−/− and ROCK2−/− mice to investigate the role of ROCK isoforms in regulating ROS production and compare the effects of the genetic approach in combination with chemical antioxidant treatments on reducing doxorubicin-induced cytotoxicity. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment, but interestingly, only ROCK1 deficiency showed greater protection than antioxidant NAC including anti-ROS production, anti-apoptotic and anti-detachment effects. In addition, only ROCK1 deficiency showed synergistic protective effects when combined with NAC. Further examinations showed that ROCK1−/− MEFs exhibited reduced NADPH oxidase activation in doxorubicin treatment, which likely contributed to the diminished ROS. Moreover, ROCK1 deficiency not only enhanced the inhibitory effects of DPI on ROS generation and caspase 3 activation, but also attenuated cell detachment caused by DPI. The superior protective effects of ROCK1 deficiency over NAC, DPI treatments or ROCK2 deficiency were associated with the preservation of central stress fibers in response to doxorubicin, which was only observed in ROCK1−/− MEFs. These results support the concept that increased actin stability from ROCK1 deficiency sustains the protective effects of antioxidants, revealing a novel strategy to enhance cytoprotective effects of antioxidant treatments against cytotoxicity induced by doxorubicin which possibly can be extended to other oxidative stresses.