Previous studies have suggested that RhoA-ROCK signaling is intricately linked to adhesion signaling. Here, by separating the effects of integrin ligation, cell spreading, and cytoskeletal tension, and directly measuring endogenous RhoA and ROCK activity, we have uncovered several important regulatory mechanisms. First, RhoA activity appears to be regulated by cell shape. Previous studies have shown that integrin-mediated adhesion can antagonize RhoA activity [11
], and are consistent with our observation of decreased RhoA activity upon addition of fibronectin-coated beads to suspended cells. This same pathway may be responsible for the further suppression of RhoA activity as cells are allowed to spread on larger micropatterned islands of fibronectin. Although we have not examined the basis for this decrease, it is plausible that the mechanism involves the increased actin polymerization observed with cell adhesion and spreading [25
]. It has been suggested that F-actin may sequester a Rho guanine-nucleotide exchange factor (Rho GEF) [26
], such that increased actin polymerization would suppress RhoA activity. Consistent with this model, we observed increased RhoA-GTP loading upon cytochalasin D treatment, a phenomenon that has been previously noted by other researchers [11
]. Additional studies will be required to further delineate this mechanism.
Second and perhaps most interestingly, the ability of RhoA to modulate ROCK kinase activity is dependent on cell adhesion, cell spreading and cytoskeletal tension. This decoupling of RhoA and ROCK is evident in suspended cells, which exhibited high levels of Rho-GTP but low MLC phosphorylation and ROCK activity. Remarkably, allowing cells to adhere on a high density of FN was not sufficient to rescue ROCK activity; instead, cell spreading and cytoskeletal tension were also required (, ). Previous studies have shown for other small GTPase effectors PAK and mDia, that integrin ligation is sufficient to mediate their activation by Rac and Rho, respectively [13
]. This decoupling of Rho and ROCK by changes in cell shape may be one important specific mechanism by which cell shape modulates cell signaling, cytoskeletal tension, and adhesion maturation. The requirement of cytoskeletal tension to couple Rho-GTP to ROCK may be mediated through the effects of mechanical tension on the maturation of focal adhesions. That is, tension mediated changes in focal adhesion maturation could affect adhesion signaling and thereby the coupling of RhoA and ROCK activity. Nonetheless, these data importantly demonstrate that GTP-RhoA activity by itself is not an accurate measure of either ROCK or contractile activity in cells (, ).
How does RhoA couple to ROCK in an adhesion dependent manner? Given that FAK is a major tyrosine kinase in focal adhesions and is important in transducing mechanical forces into biochemical signals, it may be involved in this control mechanism. FAK is required for microtubule stabilization by another RhoA effector, mDia [13
]. Tyrosine kinase activity has been shown to be required for Rho induced stress fiber formation [27
] and Rho can directly induce the phosphorylation of FAK in the absence of stress fiber formation [28
], suggesting the possibility of FAK activation being upstream of ROCK. The colocalization of RhoA and ROCK may also play a role in the regulation of ROCK activity by adhesion and tension, perhaps mediated by membrane lipid rafts, as has been shown in two other cases. Adhesion-mediated activation of both PAK by GTP-Rac and mDia by GTP-Rho have been shown to be mediated by GM1-ganglioside containing lipid rafts [13
]. The dependence of lipid raft internalization or fusion to the plasma membrane by adhesion may represent a general mechanism by which Rho GTPases and their effectors are regulated. Similar mechanisms may underlie the adhesion and cytoskeletal regulation of ROCK that we observed in this study.
The bi-directional coupling between tension and ROCK activity has numerous implications. Once activated, this positive feedback loop may be an important mechanism to amplify and sustain ROCK activity, where ROCK activates tension, which in turn strengthens the RhoA-ROCK coupling. Conversely, this architecture also explains the observation that disruption of ROCK, tension, or adhesion, functionally disrupts the entire mechano-adhesive system and results often in similar phenotypes such as decreased proliferation [2
]. It has recently been suggested that change in substrate stiffness may also affect adhesions and RhoA-ROCK coupling [3
]. Here our findings would suggest that the decreased substrate stiffness alters the mechanical stress at adhesions generated by cytoskeletal tension, in turn decreasing ROCK activity, cytoskeletal tension and adhesion maturation, and, thus providing a general control mechanism whereby cell adhesion, substrate mechanics, cytoskeletal tension, and cell signaling are all intricately related (). Since ROCK is required for generating tension, this raises the question of what generates the tension required to activate ROCK in the first place. There are other well-characterized kinases for myosin such as MLCK and PAK, that may temporally precede ROCK activation [30
]. There also exist as yet uncharacterized mechanisms for generating tension independent of MLC phosphorylation [31
] that may precede and contribute to ROCK activation.
Fig. 6 Schematic of the proposed model for how tension regulates ROCK activity. Cytoskeletal tension acts on cell-ECM adhesions to increase the signaling activity at focal adhesions. A molecular mediator at focal adhesions, allows the coupling of GTP-RhoA to (more ...)
RhoA-ROCK signaling has long been known as an important regulator of cytoskeletal tension and adhesion maturation. The numerous feedback loops between these adhesive, cytoskeletal, and mechanical functions of the cell and this signaling pathway highlight the complex control system that cells have constructed, perhaps in order to navigate the complex relationship between ECM mechanics, structure, and composition. Understanding this control system ultimately will be central to our understanding of how cells coordinate the chemical and mechanical worlds.