Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.

Abstract

Fibronectin (FN) matrix assembly is a cell-dependent process mediated by cell surfacebinding sites for the 70-kDa amino-terminal region of FN. We have shown recently that lysophosphatidic acid (LPA) is a stimulator of FN matrix assembly. Disruption of microtubules has been shown to mimic some of the intracellular effects of LPA including the formation of actin stress fibers and myosin light chain phosphorylation. We compared the effects of microtubule disruption and LPA on FN binding and actin cytoskeleton organization. The disruption of microtubules by nocodazole or vinblastine increased FN binding to adherent cells. The modulation of binding sites was rapid, dynamic, and reversible. Enhanced binding was due to increases in both the number and affinity of binding sites. These effects are similar to the effects of LPA on FN binding. Binding induced by nocodazole was inhibited by the microtubule-stabilizing agent Taxol but not by pretreatment with a concentration of phospholipase B that totally abolished the stimulatory effect of LPA. Fluorescence microscopy revealed a close correlation among actin stress fiber formation, cell contraction, and FN binding. Blockage of the small GTP binding protein Rho or actin-myosin interactions inhibited the effects of both nocodazole and LPA on FN binding. These observations demonstrate that Rho-dependent actin stress fiber formation and cell contraction induce increased FN binding and represent a rapid labile way that cells can modulate FN matrix assembly.

Cytoskeletal reorganisation, 1α,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation

“…Originally developed as a cell permeable calpain inhibitor (Tsujinaka et al, 1988), calpeptin was serendipitously reported to stimulate actin stress fiber accumulation in fibroblasts via the inhibition of Shp-2 upstream of the small GTPase Rho (Schoenwaelder and Burridge, 1999;Schoenwaelder et al, 2000). Both the naturally occurring alkaloid colchicine and the synthetic agent, nocodazole, both inhibit microtubule assembly and promote actin stress fiber accrual in a Rhodependent manner (Bershadsky et al, 1996;Enomoto et al, 1996;Zhang et al, 1997;Liu et al, 1998). As anticipated the co-treatment of MG63 osteoblast-like cells with LPA and D3 led to a robust maturation response as supported by a clear, synergistic (Fig 2B).…”

c-Cbl facilitates fibronectin matrix production by v-Abl-transformed NIH3T3 cells via activation of small GTPases

“…As indicated above, Rho-dependent cytoskeletal reorganizations have been shown by several groups to be essential for FN matrix deposition by ®broblasts (Christopher et al, 1997;Wu et al, 1995;Zhang et al, 1997;Zhong et al, 1998). This ®nding prompted us to determine whether the inhibitors that diminished deposition of FN matrix by c-Cbl-overexpressing v-Abl-transformed ®broblasts and adhesion of these cells to a non-treated surface (see Figure 6) could also inhibit spreading of these cells in culture.…”

“…Considering that Rho-dependent contractility was recently shown to be essential for the assembly of FN matrix by ®broblasts (Christopher et al, 1997;Wu et al, 1995;Zhang et al, 1997;Zhong et al, 1998), we decided to determine whether an increase in spreading of v-Abltransformed ®broblasts caused by c-Cbl overexpression facilitated FN matrix deposition by these cells through the activation of small GTPases regulating cytoskeletal rearrangements. First, we directly evaluated activities of several such GTPases in v-Abl-transformed ®bro-blasts using precipitation of their GTP-bound forms followed by speci®c immunoblotting.…”

“…The morphology of v-Abl-transformed NIH3T3 cells transduced with the control empty vector was similar to that of v-Abl-transformed NIH3T3 cells transduced to express Tyr 5 -4Phe (not shown). A representative experiment from a total of four is shown (Christopher et al, 1997;Wu et al, 1995;Zhang et al, 1997;Zhong et al, 1998), we decided to directly analyse the involvement of these GTPases in the e ects of c-Cbl in our experimental system. For these experiments we used SCH 51344, a speci®c inhibitor of Rac1-dependent signaling, and several compounds known to impede RhoA-dependent cytoskeletal reorganization; C3 exoenzyme, 3-butanedione 2-monoxime (BDM), H7 and Y27632.…”

Signaling pathways involved in OxPAPC-induced pulmonary endothelial barrier protection

“…A number of agonists including phospholipid products (Zhang, Magnusson et al 1997;Huang, Subbaiah et al 2005) and bioactive peptides (Garcia, Siflinger-Birnboim et al 1986;van Nieuw Amerongen, Draijer et al 1998;Petrache, Verin et al 2001;Moy, Blackwell et al 2002;Birukova, Adyshev et al 2005) increase vascular endothelial permeability, which my lead to alveolar flooding and pulmonary edema. However, much smaller number of bioactive molecules capable of enhancing endothelial barrier properties have been reported so far (Garcia, Liu et al 2001;Liu, Schaphorst et al 2002;VouretCraviari, Bourcier et al 2002;Birukov, Bochkov et al 2004;Kolosova, Mirzapoiazova et al 2005), and signaling mechanisms underlying these barrier protective effects have been a major focus of ongoing studies (Lee, Lee et al 2000;Garcia, Liu et al 2001;Vouret-Craviari, Bourcier et al 2002;Birukov, Leitinger et al 2004;Dudek, Jacobson et al 2004;Kolosova, Mirzapoiazova et al 2005;Mehta, Konstantoulaki et al 2005;Singleton, Dudek et al 2005).…”