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Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G(q) protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.

The Rho GTPase signaling pathway is required for actin cytoskeletal organization and serum response factor-dependent gene transcription. Lbc is a Rho-specific guanine nucleotide exchange factor that contains a modulatory C-terminal region. To elucidate Lbc regulatory mechanism(s), a yeast two-hybrid screen for proteins that interact with the Lbc C-terminal region was carried out, resulting in multiple isolation of cDNAs encoding the same 734-amino acid Lbc interacting protein. The Lbc interacting protein has homology with the alpha-catenin cell adhesion component and is identical to the alpha-catenin-like alpha-catulin protein of unknown function. The human alpha-catulin gene (CTNNAL1) maps to 9q31-32. Here we identify the predicted endogenous alpha-catulin product, document alpha-catulin and Lbc co-expression in multiple human cell lines, and show alpha-catulin and Lbc subcellular co-fractionation and intracellular localization. The required regions for Lbc and alpha-catulin interaction were mapped, and complex formation between Lbc and alpha-catulin in mammalian cells was detected. Functionally, alpha-catulin co-expression leads to increased Lbc-induced serum response factor activation in vivo as measured by a transcriptional reporter assay. Furthermore, alpha-catulin co-expression enhances Lbc-induced GTP-Rho formation in vivo. These results support the concept that the recently identified alpha-catulin protein may modulate Rho pathway signaling in vivo by providing a scaffold for the Lbc Rho guanine nucleotide exchange factor.

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106). The involvement of the G(12) family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Galpha(12) or Galpha(13) induced cell rounding, and antibodies to Galpha(12) or Galpha(13) blocked the response to thrombin. In contrast, activation or inhibition of Galpha(q) function had relatively little effect. The cytoskeletal response to Galpha(12) was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Galpha(12). The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Galpha(12) protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.

Engagement and clustering of integrins triggers a number of intracellular signaling events, including activation of the mitogen-activated protein (MAP) kinases Erk1 and Erk2. To investigate the mechanism by which integrins mediate the activation of MAP kinases upon binding of NIH 3T3 cells to fibronectin, we assessed the effects of both inhibiting and activating the small GTPase Rho. We observed that inhibition of Rho by the Rho-specific inhibitor C3 exoenzyme or by a dominant negative Rho A (RhoN19) inhibited MAP kinase activation. Conversely, activation of Rho by expression of an activated Rho A mutant (RhoQ63L), or the Rho-specific guanine nucleotide exchange factor lbc, enhanced and partially mimicked activation of Erk2 by plating on fibronectin. These results therefore show that Rho is involved in the integrin-dependent activation of MAP kinase.

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco-lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

Phenotypic abnormalities of melanocytes, germ cells, and hematopoietic cells of Steel mice demonstrate the critical role of stem cell factor (SCF) in development. Production of SCF in the hematopoietic microenvironment as either a membrane-associated or soluble factor leads to pleiotropic effects on hematopoietic stem and progenitor cells and significant effects on the production of erythroid cells. Although the production of these two forms of SCF is highly regulated, the physiologic role(s) of membrane-associated and soluble SCF remain unclear. We have demonstrated that the generation of soluble murine SCF by murine stromal cells derived from the fetal hematopoietic microenvironment is dependent on two distinct proteolytic cleavage sites. The primary site in exon 6 is preferentially utilized in these cells. The secondary site located in exon 7 is utilized only in the absence of the primary site. Proteolytic processing at this secondary site appears to be species-specific, since the human protein sequence diverges at this site, and protein expressed from the human cDNA encoding this site in murine stromal cells remains largely membrane-associated. Site-directed mutagenesis of the murine SCF cDNA encoding both proteolytic cleavage sites leads to the generation of membrane-associated and biologically active SCF on murine stromal cells. These results suggest that the regulation of processing of the secondary proteolytic cleavage site could play a critical role in the function of membrane-associated SCF protein.