Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Vascular smooth muscle ROCK1 contributes to hypoxia-induced pulmonary hypertension development in mice.

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.

Vascular cell-specific roles of mineralocorticoid receptors in pulmonary hypertension.

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients.

Glycolysis regulated transglutaminase 2 activation in cardiopulmonary fibrogenic remodeling.

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with the loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF, and type 1 diabetes that are associated with altered glucose metabolism/glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are upregulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose-induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis-mediated TG2 induction and tissue fibrosis associated with experimental PH, HF, and hyperglycemia.

Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension.

Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.

Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension.

Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.

Plasma 12- and 15-hydroxyeicosanoids are predictors of survival in pulmonary arterial hypertension.

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations. RV eicosanoids did not exhibit any changes with chronic hypoxia. PAH treatment-naïve patients had significantly increased plasma concentrations of TXB2 and 5-, 8-, 12-, and 15-HETE. The PGI2/TXB2 ratio was lower in PAH patients than in controls, especially in the treatment-naïve cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naïve, and treated patients, respectively, P = 0.001). Survival was significantly worse in PAH patients with 12-HETEhigh (≥57 pg/mL) and 15-HETEhigh (≥256 pg/mL) in unadjusted and adjusted analyses (hazard ratio [HR]: 2.8 [95% confidence interval (CI): 1.1-7.3], P = 0.04 and HR: 4.3 [95% CI: 1.6-11.8], P = 0.004, respectively; adjustment was performed with the REVEAL [Registry to Evaluate Early and Long-Term PAH Disease Management] risk score). We demonstrate significant alterations in eicosanoid pathways in experimental and human PAH. We found that 12- and 15-HETE were independent predictors of survival in human PAH, even after adjusting for the REVEAL score, suggesting their potential role as novel biomarkers.

Alpha-Catulin Co-Localizes With Vimentin Intermediate Filaments and Functions in Pulmonary Vascular Endothelial Cell Migration via ROCK.

The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role. Examination of endogenous α-catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double-immunofluorescence analyses of VEC showed endogenous α-catulin co-localization with vimentin intermediate filaments. Similar to vimentin, α-catulin was found to distribute into detergent-soluble and -insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α-catulin network distribution and altered α-catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α-catulin knock-down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α-catulin depletion was shown to reduce ROCK-induced signaling. These findings indicate that α-catulin has a unique function in co-localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. J. Cell. Physiol. 231: 934-943, 2016. © 2015 Wiley Periodicals, Inc.

Tissue transglutaminase promotes serotonin-induced AKT signaling and mitogenesis in pulmonary vascular smooth muscle cells.

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50 µM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling.

Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation.

We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.

Modulating endothelial barrier function by targeting vimentin phosphorylation.

Vimentin is a major intermediate filament protein in vascular endothelial cells which might be involved in their function as a barrier tissue. It is proposed to dynamically maintain integrity of the endothelium as a tightly regulated permeability barrier that is subjected to a variety of shear and contractile forces. The results described in this report demonstrate that vimentin plays that role through mechanisms that are dependent on its phosphorylation state. Withaferin A (WFA), a vimentin targeting drug is shown to disrupt endothelial barrier function through its effects on vimentin filament distribution and physical properties. These effects are related to WFA's ability to increase vimentin phosphorylation. Through overexpressing a non-phosphorylatable vimentin mutant we can block the effects of WFA on vimentin distribution and barrier permeability. The barrier augmentation effect appears to extend to endothelial cells that do not express detectable mutant vimentin which might suggest transmissible effects across cells. Blocking vimentin phosphorylation also protects the endothelial barrier against LPS endotoxin, implicating it as a target for drug development against pulmonary edema and acute respiratory distress syndrome (ARDS).

Elevated transglutaminase 2 activity is associated with hypoxia-induced experimental pulmonary hypertension in mice.

Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.

The Lbc Rho guanine nucleotide exchange factor α-catulin axis functions in serotonin-induced vascular smooth muscle cell mitogenesis and RhoA/ROCK activation.

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.

Serotonin induces Rho/ROCK-dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells.

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.

Alpha(E)-Catenin induces SRF-dependent transcriptional activity through its C-terminal region and is partly RhoA/ROCK-dependent.

The ubiquitous alpha(E)-catenin is an essential actin cytoskeletal linker. The transcription factor, serum response factor (SRF), induces transcription via binding to the serum response element (SRE) in gene promoters, and in many cases responds to actin dynamics. Here, we report that alpha(E)-catenin expression in HEK293 cells activates the SRE.L transcriptional reporter, a reporter containing the isolated SRF-binding site, and a stably integrated SRE.L reporter in fibroblasts. alpha-Catenin-induced reporter activity appears only partly dependent on RhoA GTPase and Rho kinase function. alpha-Catenin expression has no effect on RhoA activation or localization, and alpha-catenin-induced SRE.L reporter activation is insensitive to the actin-modulating agent latrunculin B. Ectopic alpha-catenin expression was not sufficient to induce actin filament assembly as measured by stress fiber formation. SRE.L reporter is activated by the C-terminal approximately 300 residue region of alpha(E)-catenin. These results suggest induction of SRF-mediated transcription by alpha(E)-catenin either downstream of RhoA or via a parallel pathway.

Selective inhibition of growth of tuberous sclerosis complex 2 null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mTOR/S6 kinase activity.

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.

Inhibition of serotonin-induced mitogenesis, migration, and ERK MAPK nuclear translocation in vascular smooth muscle cells by atorvastatin.

The HMG-CoA reductase inhibitors, statins, have pleiotropic effects which may include interference with the isoprenylation of Ras and Rho small GTPases. Statins have beneficial effects in animal models of pulmonary hypertension, although their mechanisms of action remain to be determined. Serotonin [5-hydroxytryptamine (5-HT)] is implicated in the process of pulmonary artery smooth muscle (PASM) remodeling as part of the pathophysiology of pulmonary hypertension. We examined the effect of atorvastatin on 5-HT-induced PASM cell responses. Atorvastatin dose dependently inhibits 5-HT-induced mitogenesis and migration of cultured bovine PASM cells. Inhibition by atorvastatin was reversed by mevalonate and geranylgeranylpyrophosphate (GGPP) supplement, suggesting that the statin targets a geranylgeranylated protein such as Rho. Concordantly, atorvastatin inhibits 5-HT-induced cellular RhoA activation, membrane localization, and Rho kinase-mediated phosphorylation of myosin phosphatase-1 subunit. Atorvastatin reduced activated RhoA-induced serum response factor-mediated reporter activity in HEK293 cells, indicating that atorvastatin inhibits Rho signaling, and this was reversed by GGPP. While 5-HT-induced ERK MAP and Akt kinase activation were unaffected by atorvastatin, 5-HT-induced ERK nuclear translocation was attenuated in a GGPP-dependent fashion. These studies suggest that atorvastatin inhibits 5-HT-induced PASM cell mitogenesis and migration through targeting isoprenylation which may, in part, attenuate the Rho pathway, a mechanism that may apply to statin effects on in vivo models of pulmonary hypertension.

Smooth muscle alpha-actin expression and myofibroblast differentiation by TGFbeta are dependent upon MK2.

Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle alpha-actin (smalpha) in response to profibrotic agents such as TGFbeta is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2(-/-)) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2(-/-) MEF to express less smalpha than their corresponding wild-type (WT) MEF at baseline and in response to TGFbeta. Furthermore, TGFbeta causes downregulation of smalpha in MK2(-/-) MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2(-/-) MEF. Our results further suggest that downregulation of smalpha in MK2(-/-) MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smalpha message stability in these cells. These results indicate that MK2 plays a key role in regulation of smalpha expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis.

Cell proliferation and drug resistance in hepatocellular carcinoma are modulated by Rho GTPase signals.

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.

Galphaz inhibits serum response factor-dependent transcription by inhibiting Rho signaling.

Galpha12/13 or Galphaq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Galphaz signals inhibit serum response factor (SRF)-dependent transcription. Galphaz expression inhibits Galpha12/13-, Galphaq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Galphaz mutants with defective fatty acylation has no inhibitory effect. Expression of Galphaz, but not Galphai, attenuates serum-induced SRE reporter activation, suggesting that Galphaz can down-regulate endogenous signals leading to SRF. Whereas Galphaz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect Galpha12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Galphaz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Galphaz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Galphaz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Galphaz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.