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Serotonin (5-HT) stimulates pulmonary artery smooth muscle cell proliferation and has been associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein receptor 2 (BMPR2) mutations similarly have been linked to PAH. However, possible crosstalk between 5-HT and BMPR signaling remains poorly characterized. We report here that 5-HT activates Smads 1/5/8 in bovine and human pulmonary artery smooth muscle cells (SMCs) and causes translocation of these Smads from cytoplasm to the nucleus. DN BMPR1A blocked 5-HT activation of Smads 1/5/8 by 5-HT and BMPR1A overexpression enhanced it. Activation of Smads by 5-HT occurred through the 5-HT 1B/1D receptor as it was blocked with the inhibitor GR 55562 but unaffected by inhibitors of the 5-HT transporter and a variety of 5-HT receptors. Activation of the Smads by 5-HT depended on Rho/Rho kinase signaling as it was blocked by Y27632, but unaffected by inhibitors of PI3K or MAPK. Transfection of cells with BMPR1A and ligation of the BMP receptor with BMP-2 also activated GTP-Rho A of these SMCs, while DN BMPR1A blocked the activation. 5-HT stimulated an increase in serine/threonine phosphorylation of BMPR1A, supporting the activation of BMPR1A by 5-HT in SMCs. Infusion of 5-HT into mice with miniosmotic infusion pumps caused activation of Smads 1/5/8 in lung tissue, demonstrating the effect in vivo. The studies support a unique concept that 5-HT transactivates the serine kinase receptor, BMPR 1A, to activate Smads 1/5/8 via Rho and Rho kinase in pulmonary artery SMCs. Rho and Rho kinase also participate in the activation of Smads by BMP.

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2-/- mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2-/- cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2-/- cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2-/- cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2-/- cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM.

Hepatocellular carcinoma is highly resistant to chemotherapeutic agents, thus the need to discover effective therapeutic molecules to suppress cancer cell growth and to overcome drug resistance is urgent. The Rho GTPase is implicated in cancer and metastasis and is directly activated by the Lymphoid blast crisis (Lbc) protooncogene, a Rho guanine-nucleotide exchange factor. The aim of the study was to analyze the expression of Lbc in hepatocarcinoma and to determine the effect of Lbc-induced Rho signaling on expression, growth rate and resistance to genotoxic stress. We found, by immunohistochemical analysis of biopsy samples and Northern and Western blot analyses of cell lines, that Lbc is absent in normal adult liver but is abundantly expressed in hepatocarcinoma, implying an increased Rho pathway signaling. Lbc stably transfected hepatocarcinoma cells exhibit increased proliferation and levels of ERK and cyclin D1 activation, which are blocked by a Rho inhibitor. In contrast, AKT activation was not altered. Moreover, Lbc expression confers increased resistance to genotoxic stress induced by doxorubicin, which is associated with upregulation of Bcl-2 and BAD phosphorylation, and this is reversed by a Rho inhibitor. In conclusion, these data support a role for Rho in liver cancer progression and resistance to therapy and may provide a basis for developing effective treatment for hepatocarcinoma.

Heterotrimeric Galpha12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Galpha signals to Rho. For example, p115 RhoGEF transduces Galpha13 signals to Rho and inhibits Galpha12/13 signals via the RhoGEF LH domain which links to Galpha subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Galpha12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Galpha12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Galpha12-induced RhoA activation in vivo. Complex formation between Galpha12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Galpha subunit signals. These findings support a role for Lbc in Galpha12-induced signaling pathways to Rho.

Cell shape changes, contractility, adhesion, migration, gene transcription, cytokinesis, membrane trafficking, and growth, require Rho small GTPase function. The basis for this is that Rho regulates actin filament assembly, and serum response factor (SRF)-mediated gene transcription. Upon activation by serum or cell adhesion, Rho stimulates a distinct signal transduction pathway that induces cytoskeletal and transcriptional responses through diverse effectors. Rho activity is tightly controlled by guanine nucleotide exchange factors, GTPase activating proteins, and guanine dissociation inhibitors. Dysregulation of the Rho pathway is implicated in multiple pathological conditions including cancer and metastasis, cardiovascular disease, bacterial and viral pathogenesis, hepatic disease, and developmental disorders.