GPCR Modulators: Statin Is Inverse Agonistic !!

3. The HMG CoA Pathway & Rho

Statins are well known inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase—the crucial rate limiting step in hepatic cholesterol biosynthesis.

Independently of the cholesterol lowering effect,
the inhibition of HMG-CoA leads to reduced synthesis of isoprenoid intermediaries—farnesyl pyrophosphate (FPP) and geranylgeranyl
pyrophosphate (GGPP)  (Figure 2) [32].

 

These precursors of cholesterol biosynthesis are essential for post-translational modification and prenylation of certain small GTPase proteins, include the Ras and Rho superfamilies [33].

Prenylation of these proteins facilitates their intracellular trafficking and covalent attachment to the lipid membrane, which is often essential for biological function.

These small GTPases cycle between an inactive GDP bound form found in the cytosol in association with guanine dissociation inhibitor (Rho GDI), and an active GTP bound form usually associated with the cell membrane (Figure 3).

When Rho proteins are released from GDIs, they can insert into the cell membrane where they are activated by guanine nucleotide exchange factors (GEFs), and this initiates interaction with membrane effector proteins such as Rho kinase (ROCK).

The Rho family of proteins is implicated in many key intracellular events and signalling pathways, including regulation of the actin cytoskeleton, cell adhesion, cell to cell interaction, and cell-cycle progression.

This topic is comprehensively reviewed by Burridge and colleagues [34].

It is thought that inhibition of the Rho pathway by statins is one of the major mechanisms via which statins affect cell physiology.

 

5.2. Rho Inhibition

 

Probably the most important pleiotropic effect of statins is the inhibition of small GTPases, including the Rho family of proteins [77].

 

The Rho pathway is responsible for various integral intracellular processes and for the interaction between cells and their environment.

 

Three subfamilies constitute the Rho superfamily—Rho, Rac, and Cdc42.

Regulation of the actin cytoskeleton, microtubule dynamics, vesicle trafficking, cell polarity and cell-cycle progression are all under the control of the Rho proteins (Figure 4).

This topic is comprehensively reviewed by Burridge et al. [34].

 

The Rho proteins contain a lipophilic isoprenoid group, which permits their attachment to the cell membrane and is generally essential for biological function.

By inhibiting the conversion of HMG-CoA to L-mevalonic acid, statins inhibit the synthesis of isoprenoid intermediaries, including FFP and GGPP.

Hence proper subcellular localisation and trafficking of these GTPase proteins is inhibited, with significant
functional consequences.

 Importantly, post-translationally immature forms of G-proteins may maintain partial function [79] [80], and interfere with the activity of mature membrane-anchored proteins.

 By inhibiting Rho and its downstream effector proteins including Rho kinase (ROCK), statins are likely to
affect the contractile properties of the conventional outflow pathway.

 

 Cells of the conventional pathway possess a contractile tone which is regulated through Rho signaling [81].

 ROCK phosphorylates and inhibits the myosin- binding subunit of myosin light chain (MLC) phosphatase.

 This action increases MLC phosphorylation and
myosin contractility, hence driving the formation of stress fibres and focal adhesion [34].

 

Early work on specific inhibitors of the Rho pathway has shown that Rho inhibition results in relaxation of the contractile tone of cells of the aqueous outflow pathway in vitro and ex vivo [78] [82] [83].

 This increases aqueous outflow and reduces intraocular pressure.

 

Indeed Rho kinase inhibitors have been shown to be potent agents in lowering intraocular pressure, and are undergoing phase 2 and 3 clinical trials [31] [84].

 This effect of Rho inhibition in lowering the O. Pokrovskaya et al. 130

 

More recent studies in this field have demonstrated the potential beneficial effect of combination therapy with a statin plus a Rho kinase inhibitor [86].

 Rho inhibitors also affect the actin cytoskeleton, and cellular morphology of the aqueous outflow pathway.

In vitro work has shown decreases in actin stress fibres and focal adhesions in cultured porcine and human TM cells [83].

 

Treatment of monkey Schlemm’s canal (SC) cells with a Rho inhibitor increases the number of giant vacuoles within cell, and decreases the expression of certain cytoskeletal proteins (ZO-1 and claudin-5) [87].

 This leads to morphological changes—cell rounding and detachment of cells from each other, as well as wider
paracellular spaces [78] [88].

 In cultured cells of the SC, Rho inhibition results in increased permeation in vitro, which facilitates aqueous drainage [88].

 Ex vivo perfusion experiments, where porcine, monkey, cow and cadaver eyes are perfused with an aqueous humour substitute, have shown that perfusion with a Rho inhibitor increases the conventional outflow facility [78] [83] [88] [89].

 

5.3. Rac and Reactive Oxygen Species (ROS)

 

One of the 3 key members of the Rho family is Rac1—this important GTPase protein is responsible for cytoskeletal
remodelling—specifically the formation of lamellipodia and membrane ruffles [90].

Lamellipodia are actin-rich cellular protrusions, essential for cell migration, and play an important role in the invasion and metastases of cancer cells [91].

 

Furthermore, Rac1 binds to p67phox which leads to activation of the NADPH oxidase
system and generation of ROS [92].

 

The presence of high concentrations of ROS can overwhelm the cell’s natural defence mechanisms and lead to programmed cell death. However the role of ROS in cell physiology is
more complex thanthat and more recent studies have shown that in some scenarios, ROS (in small doses) promote
cell survival—contrary to the traditional view that they are solely destructive molecules [93] [94].

 

ROS have also been shown to act as signalling molecules in their own right [95]. In smooth muscle and heart cells, it
has been shown that by inhibiting the prenylation and subsequent activation of Rac1, statins inactivate NADPH
oxidase and hence reduce angiotensin-II-induced ROS production [50] [96]. Our own research group has previously
demonstrated evidence of oxidative stress and mitochondrial dysfunction in lamina cribrosa cells from
the optic nerve heads of glaucoma donors, compared to normal donors [97].

 

 

Furthermore, our group has shown
that up to 50% of POAG patients have a pathogenic mitochondrial DNA mutation, which may lead to mitochondrial respiratory dysfunction and subsequent predisposition to oxidative stress in TM, LC and RGC [98].

 

Increased levels of ROS have been found in the aqueous humour of glaucoma patients [99] [100]. Glutathione is
a tripeptide found in the eye and other tissues, and is a key element of the protective mechanism of the eye
against oxidative stress [101]. Altered glutathione levels have been reported in the aqueous humour of glaucoma
patients [102], and abnormally low levels of glutathione have even been demonstrated in the serum of glaucoma
patients [101]. ROS affect the cellularity of the trabecular meshwork, and may cause endothelial dysfunction—
O. Pokrovskaya et al. 132
which would contribute to impaired aqueous outflow and higher IOP [103].

 

Mitochondrial dysfunction in LC and RG cells allows the build-up of ROS, and may lead to increased susceptibility to cell death and impaired
repair mechanisms [97] [104].

 

By reducing the production of ROS in ocular tissues, statins may help to reduce ROS-induced damage and glaucoma progression.