Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.     

RhoA and rho kinase regulate the epithelial Na+/H+ exchanger NHE3. Role of myosin light chain phosphorylation

The activity of the Na(+)/H(+) exchanger NHE3 isoform, which is found primarily in epithelial cells, is sensitive to the state of actin polymerization. Actin assembly, in turn, is controlled by members of the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore investigated the possible role of these GTPases in modulating NHE3 activity. Cells stably expressing NHE3 were transiently transfected with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity was assessed using microfluorimetry. NHE3 activity was not adversely affected by either dominant-negative Rac1 or Cdc42. By contrast, the inhibitory form of RhoA greatly depressed NHE3 activity, without noticeably altering its subcellular distribution. NHE3 activity was equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a downstream effector of RhoA, with the selective antagonist Y-27632 and a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced the phosphorylation of myosin light chain. A comparable net dephosphorylation was achieved by the myosin light chain kinase inhibitor ML9, which similarly inhibited NHE3. These data suggest that optimal NHE3 activity requires a functional RhoA-ROK signaling pathway which acts, at least partly, by controlling the phosphorylation of myosin light chain and, ultimately, the organization of the actin cytoskeleton.     

Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly

Laminin-5 is a major structural element of epithelial tissue basement membranes. In the matrix of cultured epithelial cells, laminin-5 is arranged into intricate patterns. Here we tested a hypothesis that myosin II-mediated actin contraction is necessary for the proper assembly of a laminin-5 matrix by cultured SCC12 epithelial cells. To do so, the cells were treated with ML-7, a myosin II light chain kinase inhibitor, or Y-27632, an inhibitor of Rho-kinase (ROCK), both of which block actomyosin contraction. Under these conditions, laminin-5 shows an aberrant localization in dense patches at the cell periphery. Since ROCK activity is regulated by the small GTPase Rho, this suggests that members of the Rho family of GTPases may also be important for laminin-5 matrix assembly by SCC12 cells. We confirmed this hypothesis since SCC12 cells expressing mutant proteins that inhibit RhoA, Rac, and Cdc42 assemble the same aberrant laminin-5 protein arrays as drug-treated cells. We have also evaluated the organization of the laminin-5 receptors alpha3beta1 and alpha6beta4 integrin and hemidesmosome proteins in ML-7- and Y-27632-treated cells or in cells in which RhoA, Rac, and Cdc42 activity were inhibited. In all instances, alpha3beta1 and alpha6beta4 integrin heterodimers, as well as hemidesmosome proteins, localize precisely with laminin-5 in the matrix of the cells. In summary, our results provide evidence that myosin II-mediated actin contraction and the activity of Rho GTPases are necessary for the proper organization of a laminin-5 matrix and localization of hemidesmosome protein arrays in epithelial cells.     

Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.     

Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation

Small G proteins of the Rho family are pivotal regulators of several signaling networks. The Ras homolog family (Rho) and one of its targets, Rho-associated protein kinase (ROCK), participate in a wide variety of biological processes, including bone formation. A previous study has demonstrated that the ROCK inhibitor Y-27632 enhanced bone formation induced by recombinant human bone morphogenetic protein-2 (BMP-2) in vivo and in vitro. However, the effect of other Rho family members, such as Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42), on bone formation remains unknown. In this study, we investigated whether Rac1 also participates in BMP-2-induced osteogenesis. Expression of a dominant-negative mutant of Rac1 enhanced BMP-2-induced osteoblastic differentiation in C2C12 cells, whereas a constitutively active mutant of Rac1 attenuated that effect. Knockdown of T-lymphoma invasion and metastasis 1 (Tiam1), a Rac-specific guanine nucleotide exchange factor, enhanced BMP-2-induced alkaline phosphatase activity. Further, we demonstrated that BMP-2 stimulated Rac1 activity. These results indicate that the activation of Rac1 attenuates osteoblastic differentiation in C2C12 cells.     

Involvement of G proteins of the Rho family in the regulation of Bcl-2-like protein expression and caspase 3 activation by Gastrins

Gastrins, including amidated gastrin (Gamide) and glycine-extended gastrin (Ggly), are known to accelerate the growth of gastric and colorectal cancer cells by stimulation of proliferation and inhibition of apoptosis. Gamide controls apoptosis by regulation of proteins of the Bcl-2 family and by regulation of the activation of caspases. However the interactions between Ggly and proteins of the Bcl-2 family and caspases are not known. Since in other systems G proteins of the Rho family inhibit apoptosis via interaction with proteins of the Bcl-2 family, leading to changes in caspase activities, we have compared the role of Rho family G proteins in regulation of Bcl-2-like (Bad/Bax/Bcl-xl) protein expression and caspase 3 activation by Ggly and Gamide. The effects of the specific inhibitors C3 (for Rho) and Y-27632 (for ROCK), and of dominant negative mutants of Rac, Cdc42 and PAK, were investigated in the gastric epithelial cell line IMGE-5. Apoptosis was induced by serum starvation and confirmed by annexin V staining and caspase 3 activation. Ggly inhibits caspase 3 activation via a Bcl-2-like protein-mediated pathway which requires activation of both Rho/ROCK and Rac/Cdc42/PAK. Gamide inhibits caspase 3 activation via redundant Bcl-2-like protein-mediated pathways which involve alternative activation of Rac/Cdc42/PAK and Rho/ROCK. Gamide and Ggly differentially activate members of Rho family G proteins which in turn regulate different proteins of the Bcl-2 family leading to changes in caspase 3 activity. The findings offer potential targets for blocking the growth-stimulating effects of these gastrins.     

Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.     

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

p160ROCK mediates RhoA activation of Na-H exchange.

The ubiquitously expressed Na-H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Here, stress fiber formation induced by p160ROCK was inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and was absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity was stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibited RhoA-, but not Cdc42- or Rac-activation of NEH1. In addition, the p160ROCK-specific inhibitor Y-27632 inhibited increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA, and it also inhibited LPA-increased phosphorylation of NHE1. A C-terminal truncation of NHE1 abolished both LPA-induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylated an NHE1 C-terminal fusion protein in vitro, and this was inhibited in the presence of Y-27632. Phosphopeptide maps indicated that identical residues in NHE1 were phosphorylated by p160ROCK in vivo and in vitro. These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber assembly induced by p160ROCK.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

Polarization and Migration of Hematopoietic Stem and Progenitor Cells Rely on the RhoA/ROCK I Pathway and an Active Reorganization of the Microtubule Network

Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34⁺ progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.     

Phosphatidylinositol 4-phosphate 5-kinase is essential for ROCK-mediated neurite remodeling

Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.