AGAP2: modulating TGFβ1-signalling in the regulation of liver fibrosis

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-β1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-β1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFβ1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis

Native RNA G quadruplex Immunoprecipitation (rG4IP) from mammalian cells

G quadruplex structures play an important role in regulating DNA replication and transcription and mRNA translation. Although there are several techniques that can determine its formation in vitro, the study of RNA G quadruplexes in vivo is not simple. In the current protocol, we describe an optimized technique (RNA G quadruplex immunoprecipitation [rG4IP]) to selectively pull down native cytoplasmic RNAs containing G quadruplex structures in mammalian cells. We also use a bicistronic plasmid to confirm and pinpoint the structure location. For complete details on the use and execution of this protocol, please refer to Surani et al. (2022)

Patterns of mitochondrial DNA damage in blood and brain tissues of a transgenic mouse model of Machado-Joseph disease

BACKGROUND: Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by a CAG tract expansions in the ATXN3 gene. Patterns of mitochondrial damage associated with pathological findings of brain tissues could provide molecular biomarkers of this disorder. OBJECTIVE: The potential of mitochondrial DNA (mtDNA) damage as a biomarker of MJD progression was investigated using a transgenic mouse model. METHODS: DNA was obtained from affected (pontine nuclei) and nonaffected tissues (hippocampus and blood) of transgenic animals of three distinct age groups: 8 weeks, before onset of the phenotype; 16 weeks, at onset, and 24 weeks, at well-established phenotype. Wild-type littermate mice, serving as controls, were analyzed for the same tissues and age groups. mtDNA damage was studied by fluorescence-based quantitative PCR in 84 transgenic and 93 wild-type samples. RESULTS: A clear pattern of decrease in mtDNA copy number with age and accumulation of 3,867-bp deletions at the initial stages (both being more pronounced in transgenic mice) was observed. Pontine nuclei, the affected tissue in transgenic mice, displayed 1.5 times less copies of mtDNA than nonaffected brain tissue hippocampus (odds ratio = 1.21). Pontine nuclei displayed the highest percentage of mtDNA deletions (6.05% more in transgenic mice). CONCLUSION: These results suggest that mtDNA damage is related to the initiation of the phenotype in transgenic mice; mtDNA 3,867-bp deletions may be a biomarker of the initial stages of the disease.This study was supported by the following grants: DRCT Postdoctoral fellowship to N.K. (M3.1.7/F/002/2008), FCT Postdoctoral fellowship to T.C. (SFRH/BPD/38659/2007) and C.B. (SFRH/BPD/63121/2009), FCT research grants to S.S. (PTDC/SAU-GMG/64076/2006) and A.S.F. (PIC/IC/83013/2007)

Implications of differential Transcription Start Site selection on CML and prostate cancer cell protein expression

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukaemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5’ UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumour cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role on protein expression and should not be ignored

Role of AGAP2 in the profibrogenic effects induced by TGFβ in LX-2 hepatic stellate cells

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor β 1 (TGFβ1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFβ1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFβ1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFβ1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFβ1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFβ1 induced AGAP2 promoter activation and its protein expression in LX-2. In addition, AGAP2 silencing affected TGFβ1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFβ1-induced increase of collagen-I protein levels, while its over-expression enhanced collagen I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFβ1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFβ1 signalling, contributing to the progression of hepatic fibrosis

Síndrome nefrótico y trombosis venosa extensa: causalidad o casualidad?

A case of extensive thrombosis associated to nephrotic syndrome is presented, where the diagnostic and therapeutical challenge relied on the determination of the nephropathy’s origin in this patient., which was obtained through renal biopsy that in turn suggested lupus nephritis. In this systemic pathology, as in nephrotic syndrome, a higher incidence of thromboembolic disease is found. Most adults with the nephrotic syndrome in our country have a systemic disease such as systemic lupus erithematosus, the remaining cases being usually due to primary disorders. Patients with the nephrotic syndrome have an increased incidence of arterial and venous thrombosis (particularly deep vein and renal vein thrombosis) and pulmonary emboli. In very few cases, patients whith lupus nephritis develop thrombosis in the absence of antiphospholipidic antibodies. If that were the case, it would be more related to the nephrotic syndrome, which can have different mechanisms to facilitate throembotic events, amongst which stands out the platelet activation and aggregation, the rise of VIII factor and fibrinogen levels; hypoalbuminemia and antithrombin and plasminogen activator inhibitor-1 deficit. IVC thrombosis, present in our patient, is associated with significant acute and chronic morbidity. It presents a diagnostic challenge to the clinician and requires a high index of suspicion. Thrombolysis with recombinant plasminogen tissue activator was performed, since in appropriate cases, thrombolysis is the most efficacious treatment, minimising long-term complications of IVC thrombosis.Se presenta un caso de trombosis extensa asociada a un síndrome nefrótico donde el desafío diagnóstico y terapéutico se asocia a la determinación del origen de la patología renal presente en la paciente, el cual se identificó gracias a la realización de una biopsia renal donde se encontraron hallazgos sugerentes de nefropatía lúpica. La mayoría de los adultos con síndrome nefrótico en nuestro país presentan una enfermedad sistémica como el lupus eritematoso sistémico, siendo las causas restantes de origen primario. Los pacientes con síndrome nefrótico tienen una incidencia aumentada de trombosis venosa y arterial (particularmente de trombosis venosa profunda y trombosis de las venas renales) y embolismo pulmonar. Raramente los pacientes con nefropatía lúpica desarrollan trombosis en ausencia de anticuerpos antifosfolipídicos. Si este fuera el caso, estaría más relacionado con el síndrome nefrótico, el cual puede tener diferentes mecanismos para facilitar los fenómenos trombóticos, entre los que destaca la activación y la agregación plaquetaria, la elevación del factor VIII y de los valores de fibrinógeno, la hipoalbuminemia, y la deficiencia de antitrombina y del inhibidor del activador del plasminógeno tipo 1. La trombosis de la vena cava inferior, presente en nuestra paciente, se asocia a morbilidad aguda y crónica significativa. Representa además un desafío diagnóstico para el médico internista y requiere de una alta sospecha. En nuestra pacientes se realizó trombólisis con activador tisular del plasminógeno recombinante (rTPA), ya que en los casos apropiados, la trombolisis es el tratamiento más eficaz, la cual minimiza las complicaciones a largo plazo de la trombosis de la vena cava inferior (VCI )

SP1 and RARα regulate AGAP2 expression in cancer

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is considered a proto-oncogene, but not much is known about AGAP2 gene expression regulation. To get some insight into this process, AGAP2 proximal promoter was cloned and characterised using reporter assays. We have identified SP1 as a transcription factor bound to AGAP2 promoter and required for AGAP2 expression in two different types of cancer cells (KU812, a chronic myeloid leukaemia cell line; and DU145, a prostate cancer cell line): silencing SP1 decreased AGAP2 protein levels. We have also found that all-trans retinoic acid (ATRA) treatment increased AGAP2 protein levels in both cell lines whilst curcumin treatment reduced ATRA-mediated AGAP2 increase. Furthermore, chromatin immunoprecipitation studies revealed the presence of RARα, RXRα and the lysine acetyl transferase PCAF in AGAP2 promoter. Our results provide a novel understanding of AGAP2 expression regulation that could be beneficial to those patients with cancers where AGAP2 is overexpressed

Presentación poco frecuente de la enfermedad de Takayasu. A propósito de 2 casos

Takayasu‘s disease is a chronic idiopathic inflammatory disease that mainly affects large vessels. It is predominantly seen in young women whose symptoms may appear between 15 and 30 years of age. It is characterized by a lymphomonocytic infiltrate and presence of granulomas that progress to lumen reduction, giving rise to thrombotic phenomena. In order of frequency, subclavian (85 %), descending thoracic aorta (58 %), abdominal (20 %), vertebral (17 %), iliac (16 %), and pulmonary (15 %) vessels are mainly affected. Coronary artery involvement represents less than 10 % of cases. We present two cases with unusual location, an atypical presentation involving femoral and renal arteries, and vascular involvement in a previously hypertensive pregnant woman.La enfermedad de Takayasu, es una enfermedad crónica, idiopática, inflamatoria que afecta principalmente a grandes vasos. Predomina en mujeres jóvenes cuyos síntomas pueden aparecer entre los 15 y 30 años. Se caracteriza por un infiltrado linfomonocitario y presencia de granulomas que progresan llegando hasta la reducción de la luz, desencadenando fenómenos trombóticos. En orden de frecuencia se pueden afectar las arterias subclavias (85 %), aorta torácica descendente (58 %), abdominal (20 %), vertebral (17 %), ilíaca (16 %), pulmonar (15 %) y coronarias, en menos del 10 %. Presentamos dos casos de localización poco usual y presentación atípica a nivel de vasos femorales, renales ademas de compromiso vascular en una gestante previamente hipertensa