Rho

Question 1

define RhoGTPases

Answer 1

Rho belongs to the RAS superfamily. Rho (Ras homology) Rho GTPases – family of 20 small G proteins Rho GTPases belong to the Ras superfamily of proteins: 70% homology with Ras and 85% homology with each other The best known: RhoA, Rac1 and Cdc42

Question 2

Mechanisms of activation/inactivation of RhoGTPases

Answer 2

Rho GTPases act as molecular switches: GDP-bound are inactive, GTP-bound are active ► They are activated by receptor tyrosine kinases, G protein coupled receptors, adhesion molecules, integrins, cytokines. Activating signals: G protein coupled receptor agonists, growth factors e.g. thrombin, thromboxane A2, endothelin, carbachol, angiotensin II, alpha-adrenergic agonists, sphingolipids, mechanical stress ► Their activity is regulated by 3 groups of proteins: o exchange of GDP for GTP is regulated by guanine nucleotide exchange factors (GEFs) o GTP hydrolysis is catalysed by GTPase-activating proteins (GAPs) o Inactive Rho GTPases remain in cytosol complexed with GDP dissociation inhibitors (GDIs). ► Rho GTPases are isoprenylated (geranyl-geranylated and farnesylated) and this is required for membrane localisation and activation. Membrane-bound Rho GTPases interact with downstream targets. ► Some Rho GTPases can be directly phosphorylated and this phosphorylation affects stability of complexes with GDI

Question 3

maajor processes regulated by RhoGTPases

Answer 3

• contractility
• endothelial barrier function
• motility/angiogenesis
• NADPH production of nitric oxide
• leukocyte adhesion and transmigration
• cell cycle control (proliferation)
• differentiation, apoptosis
• regulates transcription

Question 4

GTP-loading assays (Pulldown assays).

Answer 4

The levels of activated GTP-bound Rho, Rac, and Cdc42 can be measured in ‘‘pulldown assays.’’

In these assays, cell lysates are incubated with Sepharose beads with derivatized recombinant domains of target proteins of Rho, Rac, and Cdc42 (p21-activated kinase [PAK] CRIB for Rac/Cdc42, WASp-CRIB for Cdc42, rhoteckin Rho-binding domain for Rho).

As these domains only bind GTP-loaded Rho proteins, the active proteins are being literally ‘‘pulled down’’ by the beads.

the assay is then washed to remove non-bound proteins, and can undergo electrophoresis, western blotting etc

Question 5

the use of inhibitors/activators in studying Rho: statins

Answer 5

The use of inhibitors helped to elucidate the physiological importance of Rho GTPases. ► Statins inhibit cholesterol synthesis by inhibiting the rate-limiting enzyme in its pathway, HMG CoA reductase.

They also inhibit synthesis of isoprenoids, the intermediates of the cholesterol synthesis, important for the formation of lipid attachments required for the activation and membrane localisation of Rho GTPases

Question 6

► More specific inhibition /activation : mutant proteins,

Answer 6

► More specific inhibition /activation : mutant proteins, introduced into cells by microinjection or exp Next ressed following plasmid transfection or infection with viral constructs.

Created by a single amino acid substitution, dominant- negative mutants compete with endogenous GTPases for binding to cellular exchange factors (GEFs), while constitutively activated forms remain predominantly in the GTP-bound, active form

Question 7

baxterial toxins in studying Rho

Answer 7

Example:

• C3 exoenzyme ADP-ribosyl transferase from Clostridium botulinum, specifically inactivates Rho by ADP-ribosylation that impairs its activation by GEFs.
• Clostridium difficile toxins A and B inactivate all Rho family proteins by glucosylating the nucleotide binding site.
• Salmonella typhimurium SptP, acts as GAP protein for Rho family members.
• Other toxins such as cytotoxic necrotising factor from E.coli and the dermonecrotising toxin of Pertussis bacteria, activate Rho GTPases .

Question 8

Bacterial toxins and other proteins of interest can be introduced into the cells how?

Answer 8

Bacterial toxins and other proteins of interest can be introduced into the cells by cell-penetrating peptides (CPPs) derived from the TAT protein from HIV virus (TAT peptide) or Drosophila Antennapedia peptide, with less damage than the conventional techniques.

Question 9

list the Methods used to investigate the effects of Rho:

Answer 9

The use of inhibitors helped to elucidate the physiological importance of Rho GTPases

Statins ►

► More specific inhibition /activation : mutant proteins

► Use of knockout mice, cell- and tissue-specific knockouts

► Bacterial toxins: C3 exoenzyme ADP-ribosyl transferase from Clostridium botulinum, specifically inactivates Rho by ADP-ribosylation that impairs its activation by GEFs

Bacterial toxins and other proteins of interest can be introduced into the cells by cell-penetrating peptides (CPPs) derived from the TAT protein from HIV virus (TAT peptide) or Drosophila Antennapedia peptide, with less damage than the conventional techniques.

• The Rho kinase inhibitors include the pyridine derivative Y-27632, the closely related compound Y-32885 and fasudil with its derivatives

Question 10

Rho GTPases in the regulation of vascular function: examples of signalling pathways

Answer 10

Rho GTPases in the regulation of vascular function: examples of signalling pathways

 RhoA/Rho kinase pathway in the regulation of endothelial and smooth muscle cell contractility (important in the regulation of vascular tone, barrier function and cell movement/angiogenesis)

 Balance between RhoA and Rac1 important in the maintenance of endothelial barrier function

 Regulation of reactive oxygen species production (Rac1 in the NADPH oxidase activity)

 Coordinated activity of RhoA, Rac1 and Cdc42 regulates cell movement

 Cell cycle control –regulation of cell proliferation

Question 11

Rac1 effects

Answer 11

induces ruffling, lamellipodia, focal complexes + SPREADING

Question 12

Cdc42 effects:

Answer 12

induces filopodia, focal complexes, SPREADING< NAVIGATION

Question 13

RhoA induces … (brief)

Answer 13

induces stress fibres, focal adhesions, CONTRACTION

Question 14

Actin dynamics related to to what cell functional properties and which Rho?

Answer 14

cell migration, contraction, cell adhesion, cell shaape Rac1, Cdc42, RhoA

Question 15

RhoA regulates what?

Answer 15

RhoA regulates the assembly and contraction of actomyosin filaments I

n addition to activating formins to promote actin filament growth, RhoA-GTP promotes myosin-actin interactions essential for development and contraction of stress fibers, through its activation of ROCK (Rho Kinase).

ROCK increases phosphorylation of myosin II light chains. This promotes interaction of myosin with actin filaments

Question 16

Endothelial barrier function depends on…

Answer 16

the integrity of cell-to-cell juncitons

Question 17

What types of cell-to-cell junctions are there?

Answer 17

There are 3 types of endothelial junctions:

Adherens junctions (proteins: VE-cadherin, PECAM-1)

Tight junctions (occludin, claudins, JAMs)

Gap junctions (connexins 37, 40, 43)

Junctional proteins are associated with the submembrane actin cytoskeleton

Junctional integrity is regulated by phosphorylation and dephosphorylation of junctional proteins and their association with the actin cytoskeleton

Question 18

ENDOTHELIAL PERMEABILITY regulated by Rac how?

Answer 18

1. Breakdown of endothelial integrity is a consequence of increased centripetal tension created by increased actomyosin contractility and decreased intercellular adhesion (tethering forces).
2. RhoA and Rac1 have opposing effects on endothelial permability

Activation of RhoA increases actomyosin contractility, while activation of Rac1 increases tethering forces.

Question 19

Cell movement: what are the requirements, factors and steps

Answer 19

Cell movement: Coordinated activation of Rac1, Cdc42 and RhoA is required for cell movement.

1. Rac1 is essential for lamellipodia formation (forward protrusion).
2. Cdc42 is essential for filopodia formation and maintaining cell polarity (reorientation of the Golgi apparatus in the direction of movement)
3. Rac1 and Cdc42 are required for the formation of small adhesions “ focal complexes”, associated with the expanding cell front.
4. RhoA is required for maintaining cell adhesion during cell movement. It induces maturation of focal contacts into larger attachments called “focal adhesions” and is required for their turnover (detachment at the cell rear).

RhoA/Rho kinase -induced contractility at the cell rear is required for cell detachment

Question 20

Rho GTPases regulate angiogenesis and vasculogenesis. describe

Answer 20

Rho GTPases regulate angiogenesis and vasculogenesis.

Rac 1 plays a central role in endothelial cell migration, tubulogenesis, adhesion and permeability in response to vascular endothelial growth factor (VEGF).

Rac1-deficient cells are unable to migrate in response to VEGF Endothelial-specific excision of Rac1 results in embryonic lethality in mid-gestation, defective development of major blood vessels and complete lack of small vessel branching Vav2-deficient and Vav3-deficient mice (lacking GEFs for Rac1) show several cardiovascular abnormalities. Vav2-null mice display tachycardia, hypertension, and defects in the heart, arterial walls and kidneys. Vav3-deficient mice exhibit tachycardia, systemic arterial hypertension and extensive cardiovascular remodeling.

This is a combined effect of defect in the Rho protein signaling and alterations in the nervous regulation of cardiovascular functions (hyperactivity of sympathetic neurons and high catecholamine levels)

RhoA and Rac1 are highly expressed in several types of cancer (promote proliferation and metastasis)

Question 21

How does Ras affect cell cycle?

Answer 21

• Regulation of endothelial and SMC proliferation e.g. Activation of RhoA reduces levels of cell cycle inhibitors and induces translocation of kinase ERK, important in regulation of cell cycle progression

Rac1 and Cdc42 also promote G1/S transition

Question 22

Ras and ROS?

Answer 22

Rac1 is required for the assembly of NAD(P)H oxidase and Reactive Oxygen Species (ROS generation) in non-phagocytic cells ROS (O2 .-, H2O2, HO.)

impair endothelial function, inactivate endothelial NO, promote expression of pro-inflammatory genes.

This contributes to development of hypertension, atherosclerosis and other vascular disorders.

Activation of Rac1 by mechanical stress, angiotensin II and PDGF leads to ROS production in VSMCs, medial hypertrophy and endothelial senescence

Rho GTPases in Cardiovascular Diseases Atherosclerosis is a disease of the arterial wall in which the layer thickens, causing narrowing of the channel and thus, impairing blood flow. It is caused by excessive inflammatory-fibroproliferative response to numerous different forms of vascular insult. Principle cause of myocardial infarction (heart attack), stroke and gangrene of the extremities.

Question 23

RhoA implicated in several processs leading to development of atherosclerosis:

Answer 23

RhoA implicated in several processs leading to development of atherosclerosis:

1. Endothelial damage caused by oxLDL, shear stress and pro-inflammatory agents. RhoA activation results in increased in permeability, increased adhesiveness of leukocytes and platelets.
2. Leukocyte infiltration
3. SMC proliferation and migration, matrix production
4. Platelet aggregation

Question 24

Hypertension and Ras

Answer 24

Hypertension: Hallmark of hypertension – increased peripheral vascular resistance as a consequence of vascular contractility and remodelling

Evidence from animal models and patients – increased expression levels and activity of RhoA result in increased contractility and increased proliferation and migration of vascular smooth muscle cells , resulting in vascular remodelling.

Question 25

briefly summarise the actions of Rac1, RhoA, Cdc42

Answer 25

RhoA regulates the assembly of actin stress fibers,

whereas Rac1 and Cdc42 regulate actin polymerization and cellular protrusions.

Question 26

what is the pathway of actin cytoskeleton regulation?

Answer 26

Stimulation of tyrosine kinase and G protein-coupled receptors recruits and activates Rho GEFs, leading to activation of RhoA, the direct upstream activator of ROCKs.

ROCKs are pivotal downstream effectors of RhoA in regulating the actin cytoskeleton.

Question 27

In terms of cell motility, how does ROCK affect it and what happens if ROCK is inhibited?

Answer 27

Inhibitors of ROCKs such as Y-27632 and fasudil, or overexpression of dominant-negative mutants of ROCK, cause loss of actin stress fibers and focal adhesion complexes.

This is predominantly due to the phosphorylation and inhibition of MLCP by ROCKs, which increase MLC phosphorylation and cellular contraction by facilitating interaction of myosin with F-actin.

Thus ROCKs regulate cell polarity and migration through cellular contractions, protrusions, and focal adhesions.

By affecting tight and adherens junctions through actin cytoskeletal contractions, ROCKs can also regulate macrophage phagocytic activity and endothelial cell permeability.

Question 28

How do ROCK isoforms affect cardiac fibrosis and hypertrophy?

Answer 28

a recent study in haploinsufficient ROCK1/ mice indicates that ROCK1 is required for the development of cardiac fibrosis, not hypertrophy

Evidence from ROCK2_KO mouse models susggests that ROCK2 is involved in cardiomyocyte hypertrophy, apoptosis and fibrosis

Question 29

generally, what affects the bioavailability of NO?

Answer 29

Increased bioavailability of NO is, in part, dependent on increased expression and activity of eNOS as well as on decreased inactivation of NO by reactive oxygen species (ROS), specifically the superoxide anion

Question 30

what factors affect NO expression at transcriptional level?

Answer 30

various conditions and factors such as

laminar shear stress (19),

oxygen tension (62), and

TGF-1 (93)

can regulate eNOS expression at the transcriptional level

Question 31

how can NO expression be regulated at posttranscriptional level?

Answer 31

, eNOS expression can be also regulated at the posttranscriptional level.

For example, chronic hypoxia (55), TNF- (125), thrombin (25), oxidized LDL (61), and cellular proliferation are known to decrease eNOS mRNA stability.

Chronic hypoxia and cellular proliferation are known to activate RhoA and ROCKs.

In contrast, statins, which have been shown to increase eNOS mRNA stability, inhibit RhoA geranylgeranylation (58) and ROCK activity (110).

Thus RhoA/ROCK inversely regulates eNOS expression through alteration in eNOS mRNA stability.

Question 32

what are the key regulators affecting RhoA/ROCK pathway?

Answer 32

While endothelium-derived contracting factors such as Et-1 activates RhoA,

NO, the most important physiological endothelial relaxing factor, inactivates RhoA/Rock pathway.

Question 33

How does NO regulate ROCK?

Answer 33

NO is produced by endothelial NO synthase (eNOS). It then freely enters vascular smooth muscle cells and predominantly exerts its effects by stimulating the soluble guanylate cyclase and the subsequent increase in intracellular cGMP level leading to activation of cGMP-dependent protein kinase (PKG) (Fig. 2).

The serine residue at position 188 in RhoA is a PKG target downstream to NO.

PKG-mediated Ser188 phosphorylation of RhoA leads to inhibition of the RhoA/Rock signaling and a consequent reduction of the Ca2C -sensitivity of contractile proteins that plays a major role in the vasodilatory action of NO

Question 34

How does Rac1 affect RhoA?

Answer 34

Rac1/Pak1 stimulates this negative regulation of RhoA by increasing intracellular cGMP through inhibition of the type 5 phosphodiesterase (PDE5)

This mechanism could account, at least in part, for the antagonistic action of Rac1 on RhoA signaling in vascular smooth muscle cells.

In addition, NO released by the endothelial cells is itself antagonistically regulated by RhoA and Rac1

Question 35

In hypertension, what are the effects of ROCK isoforms? Evidence?

Answer 35

Rock1 haploinsufficiency or deficiency in mice affects neither basal arterial pressure nor its increase by administration of Ang II or L-NAME.

However, it leads to a reduction of hypertension-associated vascular fibrosis.

As the nonselective pharmacological inhibition of Rock1 and Rock2 isoforms that are both expressed in vascular smooth muscle cells reversed high blood pressure, it is therefore likely that Rock2 is the main isoform of Rock responsible for the vasoconstricting and hypertensive effect of RhoA signaling in mouse arteries.

Question 36

Potential role of Rac1 in HTN?

Answer 36

direct role of arterial smooth muscle cell Rac1 in the regulation of vascular smooth muscle cell contraction remains controversial, endothelial Rac1 indirectly controls arterial tone and pressure via its action on of NADPH oxidase and eNOS (Fig. 4).

In vivo, Rac1 plays a major role in the activation of NADPH oxidase and increase in oxidative stress induced by chronic Ang II administration, thereby playing an important role in the hypertrophic effect of Ang II in the cardiovascular system.

The increased oxidative stress induced by the mechanical stretch of the arterial wall produced by high blood pressure is also mediated by Rac1