Triple twist theory of Rho inhibition by the AT₂ receptor

Although there are some exceptions^1,2, numerous publications support the counter-regulatory roles of the angiotensin II (Ang II) type-2 receptor (AT₂) against the AT₁ receptor functions, such as inhibition of vascular contraction and hypertrophy^3–5. However, it is still very uncertain as to how the AT₂ receptor signals interfere with those of the AT₁ receptor in the cardiovascular system^5,6. Past findings suggest that the signal transduction of AT₁ inhibition by the AT₂ receptor may involve multiple distinct mechanisms. Some of these mechanisms appear to be indirect, such as production of nitric oxide through bradykinin opposing the vasoconstrictor actions of the AT₁ receptor³. The direct inhibitory cross-talk of the two receptors occurs proximal to the receptor hetero-dimerization, as well as downstream from the receptors between AT₁-activated protein kinases, epidermal growth factor (EGF) receptor kinase and extracellular signal-regulated kinase (ERK1/2)/p42/44 mitogen activated protein kinase (MAPK), etc, and AT₂-activated protein phosphatases, protein phosphatese 2A (PP2A), SHP-1, and MAPK phosphatase-1 (MKP-1)^7,8. The activation of the protein phosphatases by the AT₂ receptor may or may not require hetero-trimeric G proteins (G_i or G_s) and/or the recently identified AT₂ receptor C-terminal tail interacting proteins^4–6.

Given that induction of hypertrophy of vascular smooth muscle cells (VSMCs) via the AT₁ receptor appears to require a “triple-membrane-passing signal” involving a metalloprotease-dependent EGF receptor transactivation^9,10, the article by Guilluy et al in this issue of Circ Res¹¹ may not be so surprising, as it suggests the requirement of rather “twisty” three sequential phosphorylation/dephosphorylation events between a phosphatase, SHP-1, and two protein kinases for RhoA inhibition by the AT₂ receptor (see Figure 7 in the article). Although negative regulation of RhoA through its Ser188 phosphorylation by the AT₂ receptor has been demonstrated^12,13, the two kinases—caseine kinase II (CK2) and Ste20-related kinase SLK—are novel downstream elements of the AT₂ receptor.

By using multiple distinct molecular approaches, a novel signal transduction cascade for inhibition of RhoA via the AT₂ receptor, which is expected to counter-regulate RhoA activation by the AT₁ receptor in VSMCs, becomes apparent¹¹ (Figure). Rho-kinase (ROCK), the best-characterized effector of the small G protein RhoA, contributes to vascular contraction via Ca²⁺ sensitization. Moreover, the Rho/ROCK pathway has been implicated in a wide variety of cardiovascular pathogenic conditions including hypertension, atherosclerosis and cardiovascular hypertrophy^14–17. It should be noted that both heterotrimeric G protein-dependent and -independent signal transductions have been proposed to mediate AT₁ receptor function^4,18,19. In addition to the production of reactive oxygen species⁴ and enhanced VSMC contraction, hypertrophy, as well as migration induced by the AT₁ receptor, seem to require at least two parallel signal transduction cascades mediated through G_q and G_12/13. The latter is primarily implicated in the Rho/ROCK cascade activation via RGS (regulator of G protein signaling)-domain containing Rho guanine nucleotide exchange factors (RhoGEFs)^20–23. Inhibition of either cascade appears to block those pathogenic functions induced by the AT₁ receptor^20–23, and the study by Guilluy et al has further demonstrated that the RhoA inhibition mechanism via the AT₂ receptor in VSMCs results in vasodilation¹¹. The findings also indicate a strong support of this potential “triple twist” RhoA inhibition theory to explain the multiple tissue protective effects of AT₁ receptor blockers beyond the expected AT₁ inhibition, since the AT₂ receptors could be strongly stimulated under these treatments.

Figure.

Novel signal transduction cross-talk between AT₁ and AT₂ in VSMCs. The Rho/Rhokinase cascade inhibition by the AT₂ receptor via the “triple-twist” theory involving SHP-1, CK2 and SLK not only inhibits AT₁-induced vascular contraction, but also likely inhibits VSMC hypertrophy and migration. In addition, CK2 may be activated through the AT₁ receptor, and AT₂-activated SHP-1 has been shown to directly inactivate EGFR to inhibit AT₁-activated hypertrophic signal transduction.

In addition, identification of the novel key components of the AT₂ signal transduction will aid in exploring the molecular insight regarding the dynamic regulation of cardiovascular remodeling via the AT₁ versus AT₂, which likely involves far more additional cross-talk. Both cyclic AMP and cyclic GMP dependent kinases have been shown to phosphorylate RhoA at Ser188²⁴, which in part explains the vasodilatory properties of these kinases in VSMCs. The study by Guilluy et al has identified SLK as a novel RhoA Ser188 kinase¹¹. Interestingly, SLK has been shown to be able to activate apoptosis signal-regulated kinase-1 (ASK1) and p38 MAPK, leading to cell apoptosis²⁵. This fits well with the past findings that AT₂ mediates apoptosis in VSMCs via p38 MAPK activation²⁶. Regarding CK2, activation of CK2 has been recently shown to mediate p27 degradation by Ang II likely through the AT₁ receptor, which leads to cardiac hypertrophy²⁷. The p27 degradation has also been implicated in vascular hyperplasia²⁸. Therefore, inhibition of CK2 activity by the AT₂ receptor may also antagonize the AT₁ mediated detrimental effects through stabilization of p27 in addition to the RhoA inhibition.

Since the findings by Guilluy et al were mostly limited to VSMC culture and a ex vivo desendothelialized contraction assay using rat thoracic aorta rings, the relevance of this novel AT₂ signal transduction in mediating the beneficial AT₂ function in cardiovascular diseases remains unclear. Further expansion of the research in this field by using animal models of cardiovascular diseases has strong potential for future translation of the outcomes, which may lead to prevention of cardiovascular diseases linked to enhanced AT₁ receptor signal transduction.