Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Abstract

Abstract. Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA-and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPAinduced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

Dual Regulation of Actin Rearrangement through Lysophosphatidic Acid Receptor in Neuroblast Cell Lines: Actin Depolymerization by Ca2+-α-Actinin and Polymerization by Rho

“…In contrast, the R cell population reached a peak at 15-30 min and then gradually decreased ( Figure 2, d and f). The increase in the R cell population at 15 min was significantly inhibited by pretreatment with CD (Figure 3c), consistent with previous results showing that process retraction and cell rounding require actin polymerization (Jalink et al, 1994). These results indicated that LPA induced rapid actin depolymerization that was associated with loss of membrane ruffling, which was followed by actin polymerization that induced process retraction and cell rounding.…”

“…LPA-induced actin polymerization results in process retraction and cell rounding in neuronal cells such as N1E-115, or stress fiber formation in fibroblast cells (Ridley and Hall, 1992;Jalink et al, 1993). These cellular responses are mediated by actomyosin interactions through activation of the Rho pathway, produc- ing contractile forces that induce cell shape changes (Jalink et al, 1994;Tigyi et al, 1996;Kozma et al, 1997;Hirose et al, 1998;Kranenburg et al, 1999;Fukushima et al, 2000;Weiner et al, 2001). Herein, we have also shown that Rho is activated by LPA in TR cells and LPA-induced process retraction is blocked by a ROCK inhibitor or CD, which indicates the possible involvement of Rho and actomyosin for process retraction in TR cells (Figure 10).…”

Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

“…Our data show that the engagement of VE-cadherin and subsequent activation of the Rho-ROCKtension pathway are responsible for this effect in endothelial cells and support prior associations of RhoA activity with inhibition of cell spreading (Jalink et al, 1994;Hirose et al, 1998;van Leeuwen et al, 1999). Importantly, signaling through the Rho pathway also mediates the VE-cadherinstimulated increase in FAs when changes in cell spreading are prevented.…”

“…A single mechanism that might account for these paradoxical effects involves increased intracellular tension. Rho-mediated intracellular tension has been shown to decrease cell spreading (Jalink et al, 1994;Hirose et al, 1998;van Leeuwen et al, 1999), an effect that is supported by theoretical models (Palecek et al, 1999). At the same time, Rho-mediated intracellular tension has also been shown to increase stress fiber formation, integrin density, and FA formation in many studies Ballestrem et al, 2001).…”

“…To investigate whether signaling through ROCK was necessary for the VE-cadherin-mediated changes in cell spreading, we inhibited ROCK with Y-27632 in cells plated at different densities ( Figure 5B). Treatment with Y-27632 shifted the baseline to a higher degree of cell spreading even at low seeding densities, consistent with inactivation of signaling through RhoA (Jalink et al, 1994;van Leeuwen et al, 1999). Inhibiting ROCK activity also abrogated the ability of cell-cell contact to decrease cell spreading.…”