Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension

Background: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. Methods: We developed a sensitive HPLC technique to quantify dietary GGPP and conducted proteomics, qRT-PCR screening and western blot to determine signaling cascades, gene expression, protein-protein interaction and protein membrane trafficking in wild type and transgenic rats. Results: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells (HPASMCs) treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2/Rab10 signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and HPASMCs upregulated the expression of Ca 2+ -sensing receptor (CaSR) and hypoxia-induced mitogenic factor/FIZZ1 (HIMF), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10-knockdown almost abrogated hypoxia-promoted CaSR membrane-trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed with a high GGPP diet, blood GGPP levels were increased, and this abolished statin-lowering effects on plasma GGPP and hypoxia-enhanced RhoA activity of blood monocytes that were both also rescued by garlic extracts. Conclusions: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of i) RhoA-ROCK2 cascade activation and ii) Rab10-faciliated CaSR membrane trafficking with iii) subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin together with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

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The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0500 ◽

2004 ◽

Vol 15 (2) ◽

pp. 481-496 ◽

Cited By ~ 34

Author(s):

Josefa Andrade ◽

Hu Zhao ◽

Brian Titus ◽

Sandra Timm Pearce ◽

Margarida Barroso

Keyword(s):

Endoplasmic Reticulum ◽

Conformational Changes ◽

Membrane Trafficking ◽

Secretory Pathway ◽

Network Formation ◽

Binding Protein ◽

Dependent Manner ◽

Microtubule Depolymerization ◽

Independent Manner ◽

Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

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Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1140-1148.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1140-1148 ◽

Cited By ~ 37

Author(s):

Dae-Won Kim ◽

Brent H. Cochran

Keyword(s):

Map Kinase ◽

Transcriptional Activation ◽

Dominant Negative ◽

Binding Motif ◽

Dependent Manner ◽

Consensus Map ◽

Interaction Domain ◽

Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

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Abstract 17112: Transgenic Endothelial-Specific HIV-Nef Expression Induces Pulmonary Hypertension and Systemic Vascular Dysfunction, Which Can Be Reverted by Treatment With Statin

Circulation ◽

10.1161/circ.138.suppl_1.17112 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Natalia Bogatcheva ◽

Sarvesh Chelvanambi ◽

Xingjuan Chen ◽

Alexander Obukhov ◽

Matthias Clauss

Keyword(s):

Pulmonary Hypertension ◽

Vascular Dysfunction ◽

Vascular Diseases ◽

Arterial Disease ◽

Statin Treatment ◽

Systemic Circulation ◽

Vascular Pathology ◽

Systemic Hypertension ◽

Dependent Manner ◽

Specific Expression

Introduction: HIV patients on ART perplexingly remain at higher risk for developing cardiovascular diseases including acute peripheral arterial disease and pulmonary hypertension. A likely culprit for observed vascular changes is HIV protein Nef, detected both intracellularly and extracellularly in the absence of HIV RNA or DNA. Nef is known to induce endothelial dysfunction through the activation of NADPH; statins are known to inhibit NADPH activation. Hypothesis: Nef expression in endothelial cells will trigger cardiopulmonary and vascular pathology; Nef effects will be reversed by statin. Methods: Endothelial-specific expression of HIV-Nef was achieved by mating the VE-Cadherin-Tet off mice with TRE-Nef mice. The resulting Nef+ double transgenics and their Nef- negative littermates were maintained without doxycycline to induce Nef expression. Changes in pulmonary acceleration and ejection times were analyzed by ultrasound (INVEVO2100). Additionally, we assessed the ability of bradykinin-preconstricted aortic rings to dilate in response to acetylcholine in NO-dependent manner. Results: Between week 10 and week 13 of age, Nef expressing mice displayed gradual reduction of PAT/PET ratio (down to the 75% of the original PAT/PET ratio at week 10), indicative of developing pulmonary hypertension (N=6). PAT/PET ratio in Nef-negative mice did not change significantly between week 10 and 13 of age. Importantly, statin treatment initiated at week 10 completely suppressed PAT/PET changes developing in Nef-expressing mice. Arterial rings from Nef expressing mice (n=4) showed significantly impaired dilatation in response to acetylcholine (10% relaxation in Nef+ mice vs 40% relaxation in Nef-negative littermates, p=0.03), indicative of changes in systemic circulation. This difference was significantly attenuated in Nef+ mice receiving statin treatment. Conclusions: Our data suggests that mice with endothelial expression of HIV-Nef display pathological changes in pulmonary and systemic circulation. Statin treatment significantly attenuates changes in parameters indicative of pulmonary and systemic hypertension, suggesting that statin will be beneficial for patients with HIV-induced cardiopulmonary and vascular diseases.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Diagnosis and clinical investigation of patients presenting with pulmonary hypertension

ESC CardioMed ◽

10.1093/med/9780198784906.003.0584 ◽

2018 ◽

pp. 2507-2511 ◽

Cited By ~ 1

Author(s):

Daniela Calderaro ◽

Luis Felipe Prada ◽

Rogério Souza

Keyword(s):

Pulmonary Hypertension ◽

Clinical Investigation ◽

Right Heart Catheterization ◽

Heart Catheterization ◽

Right Heart ◽

Reference Centre ◽

Mean Pulmonary Arterial Pressure ◽

Invasive Test ◽

Pulmonary Arterial

The diagnosis of pulmonary hypertension (PH) relies on the haemodynamic criterion of mean pulmonary arterial pressure greater than or equal to 25 mmHg, assessed by right heart catheterization. The scope of this chapter is to discuss the key elements of clinical assessment of PH patients and the decision process to indicate right heart catheterization. Investigation must get through all the possible causes of PH according to their probability and frequency in the population. Echocardiography is the most important non-invasive test as an indicator for further diagnostic evaluation. Patients who are eligible for right heart catheterization should always be referred to PH centres, where technical skills and standardized procedures will enable maximal reliability of haemodynamic measurement. In the reference centre, a multidisciplinary team will discuss clinical and haemodynamic data, to propose the best therapeutic and follow-up schedule.

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Statin therapy in pulmonary hypertension

PVRI Review ◽

10.4103/0974-6013.57749 ◽

2009 ◽

Vol 1 (4) ◽

pp. 199 ◽

Cited By ~ 1

Author(s):

RedaE Girgis

Keyword(s):

Pulmonary Hypertension ◽

Statin Therapy

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Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses

Cells ◽

10.3390/cells8101146 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1146 ◽

Cited By ~ 4

Author(s):

Brown ◽

del Corsso ◽

Zoidl ◽

Donaldson ◽

Spray ◽

...

Keyword(s):

Protein Transport ◽

Mutational Analysis ◽

Binding Motif ◽

Dependent Manner ◽

Electrical Synapses ◽

Binding Motifs ◽

Connexin 36 ◽

Dependent Transport ◽

Carboxy Terminal

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B–microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

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Transcriptional Coactivator TAZ Negatively Regulates Tumor Suppressor p53 Activity and Cellular Senescence

Cells ◽

10.3390/cells9010171 ◽

2020 ◽

Vol 9 (1) ◽

pp. 171

Author(s):

Chiharu Miyajima ◽

Yuki Kawarada ◽

Yasumichi Inoue ◽

Chiaki Suzuki ◽

Kana Mitamura ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Suppressor ◽

Cellular Senescence ◽

Hippo Pathway ◽

Physiological Role ◽

Underlying Mechanism ◽

Binding Motif ◽

Dependent Manner ◽

Transcriptional Coactivator ◽

Tumor Suppressor P53

Transcriptional coactivator with a PDZ-binding motif (TAZ) is one of the mammalian orthologs of Drosophila Yorkie, a transcriptional coactivator of the Hippo pathway. TAZ has been suggested to function as a regulator that modulates the expression of cell proliferation and anti-apoptotic genes in order to stimulate cell proliferation. TAZ has also been associated with a poor prognosis in several cancers, including breast cancer. However, the physiological role of TAZ in tumorigenesis remains unclear. We herein demonstrated that TAZ negatively regulated the activity of the tumor suppressor p53. The overexpression of TAZ down-regulated p53 transcriptional activity and its downstream gene expression. In contrast, TAZ knockdown up-regulated p21 expression induced by p53 activation. Regarding the underlying mechanism, TAZ inhibited the interaction between p53 and p300 and suppressed the p300-mediated acetylation of p53. Furthermore, TAZ knockdown induced cellular senescence in a p53-dependent manner. These results suggest that TAZ negatively regulates the tumor suppressor functions of p53 and attenuates p53-mediated cellular senescence.

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Promotion of osteoclastogenesis by IL-26 in rheumatoid arthritis

Arthritis Research & Therapy ◽

10.1186/s13075-019-2070-0 ◽

2019 ◽

Vol 21 (1) ◽

Author(s):

Kyung-Ann Lee ◽

Kyoung-Woon Kim ◽

Bo-Mi Kim ◽

Ji-Yeon Won ◽

Hong Ki Min ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Peripheral Blood ◽

Osteoclast Differentiation ◽

Joint Damage ◽

Enzyme Linked Immunosorbent Assay ◽

Tartrate Resistant Acid Phosphatase ◽

Dependent Manner ◽

Receptor Subunit ◽

Blood Monocytes ◽

Peripheral Blood Monocytes

Abstract Background The inflammatory cascade in the rheumatoid arthritis (RA) synovium is modulated by a variety of cytokine and chemokine networks; however, the roles of IL-26, in RA pathogenesis, are poorly defined. Here, we investigated the functional role of interleukin-26 (IL)-26 in osteoclastogenesis in RA. Methods We analyzed levels of IL-20 receptor subunit A (IL-20RA), CD55, and receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL) in RA fibroblast-like synoviocytes (FLSs) using confocal microscopy. Recombinant human IL-26-induced RANKL expression in RA-FLSs was examined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured with macrophage colony-stimulating factor (M-CSF) and IL-26, after which osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells. Additionally, osteoclastogenesis was evaluated by monocytes co-cultured with IL-26-prestimulated FLSs. Results The expression of IL-20RA in RA-FLSs was higher than that in osteoarthritis-FLSs. Additionally, in IL-26-pretreated RA-FLSs, the expression of IL-20RA (but not IL-10 receptor subunit B) and RANKL increased in a dose-dependent manner, with IL-26-induced RANKL expression reduced by IL-20RA knockdown. Moreover, IL-26-induced RANKL expression was significantly downregulated by inhibition of signal transducer and activator of transcription 1, mitogen-activated protein kinase, and NF-κB signaling. Furthermore, IL-26 promoted osteoclast differentiation from peripheral blood monocytes in the presence of low dose of RANKL, with IL-26 exerting an additive effect. Furthermore, co-culture of IL-26-pretreated RA-FLSs with peripheral blood monocytes also increased osteoclast differentiation in the absence of addition of RANKL. Conclusions IL-26 regulated osteoclastogenesis in RA through increased RANKL expression in FLSs and direct stimulation of osteoclast differentiation. These results suggest the IL-26/IL-20RA/RANKL axis as a potential therapeutic target for addressing RA-related joint damage.

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Regulation of the endosomal SNX27-retromer by OTULIN

Nature Communications ◽

10.1038/s41467-019-12309-z ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Aurelia Stangl ◽

Paul R. Elliott ◽

Adan Pinto-Fernandez ◽

Sarah Bonham ◽

Luke Harrison ◽

...

Keyword(s):

Cell Surface ◽

Membrane Trafficking ◽

Pdz Domain ◽

Binding Motif ◽

Ubiquitin Chain ◽

Sorting Nexin ◽

Catalytic Function ◽

Retromer Complex ◽

Mass Spectrometric Identification ◽

Affinity Interaction

Abstract OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

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