Rho-Rock Signaling Pathway

Introduction

Rho GTPase belongs to the Ras superfamily and is involved in cell migration, phagocytosis, contraction, and adhesion. ROCK, also known as a Rho-associated kinase, is the most detailed Rho downstream target effector in functional studies. The Rho/ROCK signaling pathway induces cytoskeletal reorganization, cell migration, and stress fiber formation, and is involved in various physiological functions such as endothelial permeability, tissue contraction, and growth. It is also involved in the development of diseases such as diabetic nephropathy, eye diseases, tumors, heart disease, nerve damage diseases, hypertension, radiation damage, and leukemia, and has attracted more and more attention as a drug development target.

Basic biological characteristics of Rho-ROCK signaling pathway

The Rho GTPase members found in mammalian tissue cells are mainly Rho (A, B, C), Rac (1, 2, 3), Cdc42 (Cdc42Hs/G25K, TC10, Tcl), RhoD, RhoG, Chp (1, 2), Rnd (RhoE/Rnd3, Rnd1/Rho6, Rnd2/Rho7), RhoH/TTF, Rif, Wrch1 and RhoBTB (1, 2). The most studied types are Rho, Rac, and Cdc42. Rho GTPase is involved in activities such as cell migration, phagocytosis, contraction, and adhesion. Among them, Rho can promote stress fiber formation and elongation, actin bundle contraction and directional adhesion; Rac and Cdc42 mainly induce the formation of lamellipodia and silk feet and promote prominent activities.

ROCK, also known as Rho-associated kinase, belongs to the serine/threonine protein kinase and has a molecular mass of about 160 kD. It is the most detailed Rho downstream target effector molecule in functional research. ROCK has many functions such as regulating cell contraction, migration, adhesion and proliferation. Pharmacological studies have found that the occurrence of various diseases such as diabetic nephropathy, cancer, hypertension, nerve damage and glaucoma are related to ROCK. The ROCK amino acid sequence consists of a kinase domain (RBD) (amino terminus), a coiled-coil region, a PH domain (pleckstrin homology PH), and a cysteine-rich domain (CRD) (carboxy terminal), in which the Rho-binding domain (RBD) is in the spiral helix domain. ROCK includes the ROCK1 (ROKβ, p160-ROCK) and ROCK2 (ROKα) isoforms, which have an amino acid sequence identity of 65% and a high degree of similarity in the kinase domain (92% consensus). ROCK is distributed throughout the body. In comparison, ROCK1 is more highly expressed in non-neural tissues (liver, lung, spleen, and testis), while ROCK2 is more expressed in brain, heart, and muscle. Gene silencing experiments showed that ROCK1 plays a key role in stress fiber formation, and ROCK2 plays an important role in phagocytosis and cell contraction.

Rho has two states: inactivation combined with GDP and activation with GTP. The relative proportions of the two states are regulated by GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF). The Rho/ROCK signaling pathway activates downstream ROCK by Rho binding to GTP and further phosphorylates ROCK downstream substrates, remodeling the cytoskeleton, inducing actin filament stabilization and actin-myosin contraction, combing actin networks and myosin fibers, regulating microtubule dynamics. The main downstream substrate of ROCK and its phosphorylated effects are shown in Figure 1. The main substances include Myosin light chain (MLC), Myosin light chain phosphatase (MLCP), ERM (Ezrin/Radixin/Moesin), Adducin, LIM-kinase (LIMK), CPI-17, CRMP-2 (DPYL2), MAP-2/Tau, Intermediate filaments (GFAP, Vimentin), Par3, eNOS, etc.

Figure 1 Regulation, functions, and inhibition of the Rho-ROCK-controlled cellular processes

The physiological role of Rho-ROCK signaling pathway

RhoA is activated when stimulated by histamine, thrombin, vascular endothelial growth factor, lipopolysaccharide, and mechanical action. The combination of activated RhoA and ROCK increases calmodulin formation, up-regulates intracellular Ca2+ concentration, activates myosin light chain kinase (MLCK), and up-regulates the level of phospho-myosin light Chain (p-MLC). At the same time, phosphorylation of myosin light chain phosphatase (MLCP) inhibits dephosphorylation of p-MLC, resulting in increased permeability of the vascular endothelium and attenuated barrier function. The effect of ROCK on endothelial permeability is different from that of vascular endothelium. ROCK protects the integrity of the lymphatic endothelial barrier and reduces the increased permeability of the lymphatic endothelium when stimulated by histamine and thrombin. In addition, Rho plays an important role in the regulation of the actin skeleton, which regulates E-cadherin-mediated intercellular adhesion junctions and affects epithelial tissue function. E-cadherin induces Rac activation, and initial Rac activation causes p190RhoGAP activation to inhibit RhoA activity, and Rac activity is gradually replaced by RhoA to form a mature intercellular adhesion junction. When stimulated by alcohol, intestinal Rho-ROCK signaling pathway is activated, leading to increased intestinal permeability and impaired barrier function. Rho/ROCK signaling pathway activation can break down tight junctions and participate in TGF-β1-induced renal tubular epithelial-mesenchymal transdifferentiation.

Infection-induced neonatal preterm birth is associated with activation of the Rho-ROCK signaling pathway. In vitro studies have shown that ROCK inhibitors can significantly attenuate myometrial cell contraction induced by lipopolysaccharide stimulation. ROCK2 regulates the directional movement of myoblasts by regulating focal adhesion formation and maturation and plays an important role in skeletal muscle growth and regeneration. When ROCK2 is inhibited, the migration rate of myoblasts is accelerated, and the directionality is weakened, which inhibits the maturity of adhesion spots. Inhibition of the Rho-ROCK signaling pathway inhibits glial growth inhibitory factor activity and promotes the growth of damaged axons. ROCK2 is the dominant subtype in this process. The Rho-ROCK signaling pathway is involved in the permeability of the bone marrow mesenchymal stem cells through the blood-brain barrier, which inhibits the transmembrane transport of cells.

Rho-ROCK signaling pathways and diseases

Heart disease

Activation of RhoA-ROCK signaling pathway can increase the level of local ischemic myocardial fibrosis, and the expression of RhoA and ROCK in cardiac tissue of rats with acute myocardial fibrosis is significantly increased. The Rho-ROCK signaling pathway is involved in NAD(P)H oxidase activation, induces oxidative stress, cardiac microvascular injury, and C-reactive protein-induced atherosclerotic thrombosis. C-reactive protein also increases NF-κB activity through activation of this signaling pathway, up-regulating the transcription and expression of the atherothrombotic gene PAI-1. High glucose can activate the Rho-ROCK pathway, induce the expression of visceral adipokines and type I procollagen in cardiomyocytes, and trigger the proliferation of cardiomyocytes resulting in diabetic cardiomyopathy.

Figure 2 Role of ROCK1 and ROCK2 in cardiovascular disease

ROCK inhibitors can improve vascular smooth muscle cell over-contraction, endothelial dysfunction, inflammatory cell infiltration, vascular and myocardial remodeling, and protect the heart. Statins reduce serum cholesterol levels, increase endothelial function, and reduce vascular inflammatory responses to treat atherosclerosis, which is associated with inhibition of the Rho/ROCK signaling pathway.

Eye disease

In most retinal diseases, the retinal-blood barrier is impaired, allowing thrombin in the blood to come into direct contact with the retinal pigment epithelium. Thrombin, in turn, activates the Rho-ROCK signaling pathway to promote actin stress fiber formation and MLC phosphorylation, inducing retinal pigment epithelial cell transformation and migration, leading to retinal-blood barrier dysfunction. High glucose can affect the barrier function of microvascular endothelial cells and induce diabetic retinopathy by activating the Rho-ROCK signaling pathway. The Rho-ROCK signaling pathway is involved in the occurrence of glaucoma, which inhibits the pathway, thereby promoting relaxation of the trabecular meshwork, increasing aqueous humor flow, and reducing intraocular pressure. It also inhibits the transdifferentiation of the fascial sac fibroblasts into myofibroblasts; protects the ocular nerves and increases blood flow; increases retinal ganglion cell survival and axonal regeneration.

Tumor

The effect of Rho/ROCK signaling pathway on biofilm permeability affects the metastasis of cancer cells. Inhibition of ROCK can reduce the invasion and metastasis of tumor cells such as lung cancer, breast cancer, liver cancer, gastric cancer, and abdominal aneurysms. The Ras/Rho/ROCK signaling pathway can affect the development of lysophospho-induced ovarian cancer by affecting the secretion of proteolytic enzymes. When the DEK proto-oncogene of human non-small cell lung cancer is depleted, the expression of key proteins in the RhoA/ROCK signaling pathway is significantly reduced.

References:

  1. Marie, M. F., Wewer, U. M., & Atsuko, Y. (2013). Regulation of rock activity in cancer. Journal of Histochemistry & Cytochemistry, 61(3), 185-198.
  2. Yu, Y., Qin, J., Liu, M., Ruan, Q., Li, Y., & Zhang, Z. (2014). Role of rho kinase in lysophosphatidic acid-induced altering of blood-brain barrier permeability. International Journal of Molecular Medicine, 33(3), 661.
  3. Amin, E., Dubey, B. N., Zhang, S. C., Gremer, L., Dvorsky, R., & Moll, J. M., et al. (2013). Rho-kinase: regulation, (dys)function, and inhibition. Biological Chemistry, 394(11), 1399-1410.
  4. Nakahara, S., Tsutsumi, K., Zuinen, T., & Ohta, Y. (2015). Filgap, a rho-rock-regulated gap for rac, controls adherens junctions in mdck cells. Journal of Cell Science, 128(11), 2047-56.
  5. Elamin, E., Masclee, A., Dekker, J., & Jonkers, D. (2014). Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated rho/rock activation. American Journal of Physiology Gastrointestinal & Liver Physiology, 306(8), 677-85.
  6. Zhang, K., Zhang, H., Xiang, H., Liu, J., Liu, Y., & Zhang, X., et al. (2013). Tgf-β1 induces the dissolution of tight junctions in human renal proximal tubular cells: role of the rhoa/rock signaling pathway. International Journal of Molecular Medicine, 32(2), 464-468.
  7. Hutchinson, J. L., Rajagopal, S. P., Yuan, M., & Norman, J. E. (2014). Lipopolysaccharide promotes contraction of uterine myocytes via activation of rho/rock signaling pathways. Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology, 28(1), 94.
  8. Goetsch, K. P., Snyman, C., Myburgh, K. H., & Niesler, C. U. (2014). Rock-2 is associated with focal adhesion maturation during myoblast migration. Journal of Cellular Biochemistry, 115(7), 1299-1307.
  9. Roloff, F., Scheiblich, H., Dewitz, C., Dempewolf, S., Stern, M., & Bicker, G. (2015). Enhanced neurite outgrowth of human model (nt2) neurons by small-molecule inhibitors of rho/rock signaling. Plos One, 10(2), e0118536.
  10. Koch, J. C., Tönges, L., Barski, E., Michel, U., Bähr, M., & Lingor, P. (2014). Rock2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the cns. Cell Death & Disease, 5(3), e1225.
  11. Hartmann, S., Ridley, A. J., & Lutz, S. (2015). The function of rho-associated kinases rock1 and rock2 in the pathogenesis of cardiovascular disease. Frontiers in Pharmacology, 6(Suppl. 1).

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