Michael F. Olson

An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1

Members of the Rho family of small guanosine triphosphatases (GTPases) regulate the organization of the actin cytoskeleton; Rho controls the assembly of actin stress fibers and focal adhesion complexes, Rac regulates actin filament accumulation at the plasma membrane to produce lamellipodia and membrane ruffles, and Cdc42 stimulates the formation of filopodia. When microinjected into quiescent fibroblasts, Rho, Rac, and Cdc42 stimulated cell cycle progression through G1 and subsequent DNA synthesis. Furthermore, microinjection of dominant negative forms of Rac and Cdc42 or of the Rho inhibitor C3 transferase blocked serum-induced DNA synthesis. Unlike Ras, none of the Rho GTPases activated the mitogen-activated protein kinase (MAPK) cascade that contains the protein kinases c-Raf1, MEK (MAPK or ERK kinase), and ERK (extracellular signal-regulated kinase). Instead, Rac and Cdc42, but not Rho, stimulated a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun NH2-terminal kinase or stress-activated protein kinase). Rho, Rac, and Cdc42 control signal transduction pathways that are essential for cell growth.

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin–myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin–myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin–myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1.

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function,. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.

Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility.

Transformation by oncogenic Ras requires the function of the Rho family GTPases. We find that Ras‐transformed cells have elevated levels of RhoA‐GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho‐GTP are not a direct consequence of Ras signalling but are selected for in response to sustained ERK–MAP kinase signalling. While the elevated levels of Rho‐GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibres in transformed cells. We show that the sustained ERK–MAP kinase signalling resulting from transformation by oncogenic Ras down‐regulates ROCK1 and Rho‐kinase, two Rho effectors required for actin stress fibre formation. The repression of Rho‐ dependent stress fibre formation by ERK–MAP kinase signalling contributes to the increased motility of Ras‐transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibres and inhibits the motility of Ras‐transformed fibroblasts. We propose a model in which Ras and Rho signalling pathways cross‐talk to promote signalling pathways favouring transformation.

RAS and RHO GTPases in G1-phase cell-cycle regulation

As RAS mutations are among the most frequent alterations in human cancers, RAS proteins and their signalling pathways have been studied intensively. Here, we outline the contributions of H-RAS, N-RAS and K-RAS to cell-cycle progression and cell growth. We also summarize recent results that indicate how other members of the RAS-GTPase subfamily — including E-RAS, RHEB, R-RAS, TC21 and RAL, as well as RHO GTPases — promote proliferation by regulating the transcription, translation and degradation of key cell-cycle components.

Actomyosin-Mediated Cellular Tension Drives Increased Tissue Stiffness and β-Catenin Activation to Induce Epidermal Hyperplasia and Tumor Growth

Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. β-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters.

Applications for ROCK kinase inhibition

ROCK kinases, which play central roles in the organization of the actin cytoskeleton, are tantalizing targets for the treatment of human diseases. Deletion of ROCK I in mice revealed a role in the pathophysiological responses to high blood pressure, and validated ROCK inhibition for the treatment of specific types of cardiovascular disease. To date, the only ROCK inhibitor employed clinically in humans is fasudil, which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm. Clinical trials, mostly focusing on the cardiovascular system, have uncovered beneficial effects of fasudil for additional indications. Intriguing recent findings also suggest significant potential for ROCK inhibitors in the production and implantation of stem cells for disease therapies.

Rho GTPase signalling pathways in the morphological changes associated with apoptosis

The killing and removal of superfluous cells is an important step during embryonic development, tissue homeostasis, wound repair and the resolution of inflammation. A specific sequence of biochemical events leads to a form of cell death termed apoptosis, and ultimately to the disassembly of the dead cell for phagocytosis. Dynamic rearrangements of the actin cytoskeleton are central to the morphological changes observed both in apoptosis and phagocytosis. Recent research has highlighted the importance of Rho GTPase signalling pathways to these changes in cellular architecture. In this review, we will discuss how these signal transduction pathways affect the structure of the actin cytoskeleton and allow for the efficient clearance of apoptotic cells.

Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases

BACKGROUND Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation. RESULTS Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. CONCLUSIONS We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

LIM kinases : function, regulation and association with human disease

The LIM kinase family consists of just two members: LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2). With uniquely organised signalling domains, LIM kinases are regulated by several upstream signalling pathways, principally acting downstream of Rho GTPases to influence the architecture of the actin cytoskeleton by regulating the activity of the cofilin family proteins cofilin1, cofilin2 and destrin. Although the LIM kinases are very homologous, particularly when comparing kinase domains, there is emerging evidence that each may be subject to different regulatory pathways and may contribute to both distinct and overlapping cellular and developmental functions. Normal central nervous system development is reliant upon the presence of LIMK1, and its deletion has been implicated in the development of the human genetic disorder Williams syndrome. Normal testis development, on the other hand, is disrupted by the deletion of LIMK2. In addition, the possible involvement of each kinase in cardiovascular disorders as well as cancer has recently emerged. The LIM kinases have been proposed to play an important role in tumour-cell invasion and metastasis; fine-tuning the balance between phosphorylated and non-phosphorylated cofilin may be a significant determinant of tumour-cell metastatic potential. In this review, we outline the structure, regulation and function of LIM kinases and their functions at cellular and organismal levels, as well as their possible contributions to human disease.