RAP1B Research Articles

STRENGTHENING THE EVIDENCE: RAP1B GENE ASSOCIATION WITH SYNDROMIC THROMBOCYTOPENIA

STRENGTHENING THE EVIDENCE: RAP1B GENE ASSOCIATION WITH SYNDROMIC THROMBOCYTOPENIA

Abstract P2084: Determining The Role Of Rap1b In Megakaryocyte Survival

Platelet counts are tightly regulated through production by megakaryocytes and clearance by hepatocytes. Thrombotic risk and poor cancer prognosis correlate with platelet counts on the high and low ends of the normal range, respectively. Therefore, understanding the factors that regulate megakaryocyte survival and function are important for human health. The small GTPase, Rap1b increases in abundance as megakaryocytes mature and mediates survival in other cell types by preventing apoptosis. We hypothesize that disruption of the Rap1b gene in the megakaryocytic leukemia cell line, DAMI, will limit cell survival by increasing susceptibility to apoptosis. We used CRISPR/Cas to specifically disrupt the Rap1b gene, resulting in no detectable Rap1b protein in DAMI cells. Compared to wild-type DAMI cells, Rap1b knockout cells had nearly a 50% reduction in survival as measured by metabolic activity using an MTT-assay. To assess apoptosis susceptibility, we monitored mitochondrial membrane potential using the fluorescent sensor JC-1 and caspases activity using the Fluorochrome Inhibitor of Caspases (FLICA). These results suggest that Rap1b promotes megakaryocyte survival.

1,800 MHz Radiofrequency Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells.

Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development.Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway.Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells.Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF-EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h.Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus.

Prognostic and immunological role of Ras-related protein Rap1b in pan-cancer

ABSTRACT Ras-related Protein Rap1b, a GTP-binding protein belonging to the proximal RAS, which affects tumor progression through regulating tumor cell proliferation, invasion and participates in the functions of various immune cells. However, the potential roles and mechanisms of Rap1b in tumor progression and immunology remains unclear. In this study, we systematically analyzed the pan-cancer expression and prognostic correlation of Rap1b based on GTEX, CCLE, Oncomine, PrognoScan, Kaplan–Meier plotters and TCGA databases. The potential correlations of Rap1b with immune infiltration were revealed via TIMER and TCGA database. SangerBox database was used to analyzed the correlations between Rap1b expression and immune checkpoint (ICP), tumor mutational burden (TMB), microsatellite instability (MSI), mismatch repairs (MMRs) and DNA methylation. The results indicated that the expression level of Rap1b varies in different tumors. Meanwhile, the expression level of Rap1b strongly correlated with prognosis in patients with tumors, higher expression of Rap1b usually was linked to poor prognosis in different datasets. Rap1b was correlated closely with tumor immunity and interacted with various immune cells in different types of cancers. In addition, there were significant positive correlations between Rap1b expression and ICP, TMB, MSI, MMRs and DNA methylation. In conclusion, the results of pan-cancer analysis showed that the abnormal Rap1b expression was related to poor prognosis and tumor immune infiltration in different cancers. Furthermore, Rap1b gene may be used as a potential biomarker of clinical tumor prognosis.

De novo missense variants in the RAP1B gene identified in two patients with syndromic thrombocytopenia.

We present two independent cases of syndromic thrombocytopenia with multiple malformations, microcephaly, learning difficulties, dysmorphism and other features. Exome sequencing identified two novel de novo heterozygous variants in these patients, c.35G>T p.(Gly12Val) and c.178G>C p.(Gly60Arg), in the RAP1B gene (NM_001010942.2). These variants have not been described previously as germline variants, however functional studies in literature strongly suggest a clinical implication of these two activating hot spot positions. We hypothesize that pathogenic missense variants in the RAP1B gene cause congenital syndromic thrombocytopenia with a spectrum of associated malformations and dysmorphism, possibly through a gain of function mechanism.

MicroRNA-149 inhibits the progression of lung adenocarcinoma through targeting RAP1B and inactivating Wnt/β-catenin pathway.

MicroRNAs (miRNAs) are important regulators in the progression of lung adenocarcinoma (LAD). Moreover, microRNA-149 (miR-149) exhibits different roles in human cancers. Hence, this study mainly focused on the function of miR-149 in LAD. Western blot analysis and Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) were used to quantify expression levels. The regulatory mechanism of miR-149/RAP1B was explored by methyl thiazolyl tetrazolium (MTT), transwell, and Dual-Luciferase reporter assays. Downregulation of miR-149 was detected in LAD and predicted worse prognosis in patients with LAD. Functionally, overexpression of miR-149 inhibited cell viability and metastasis in LAD. In addition, miR-149 directly targets RAP1B and restrained its expression in LAD. Furthermore, upregulation of RAP1B attenuated the inhibitory effect of miR-149 on LAD. Besides that, miR-149 blocked epithelial-mesenchymal transition (EMT) and Wnt/β-catenin pathway in LAD. MiR-149 inhibited the progression of LAD by restraining RAP1B/EMT and inactivating Wnt/β-catenin pathway.

The balance of life and death may hinge on the intracellular localization of the small GTPase, Rap1b in the megakaryocyte cell line (DAMI)

Platelets are specialized cell fragments that circulate in the bloodstream and clot to prevent blood from leaving damaged vessels. The number of circulating platelets is governed by the balance of platelet production by mature megakaryocytes and clearance of platelets from the bloodstream. Normal platelet function requires the signaling protein, Rap1b, whose abundance also increases as megakaryocytes mature. The intracellular location of Rap1b is determine by its phosphorylation state. When Rap1b is not phosphorylated, it associates with membranes; conversely when Rap1b is phosphorylated, it detaches from membranes and reside in the cytosol. We hypothesize that Rap1b, specifically in its phosphorylated, cytosolic state, promotes megakaryocytes survival and proliferation. To address this hypothesis, the human megakaryocyte cell line (DAMI cells) have been modified 1) by disrupt the rap1b gene creating a Rap1b‐knockout cell line, 2) stable overexpression of a Rap1b phospho‐mimetic mutant, 3) stable overexpression of a Rap1b phospho‐deficient mutant or 4) stable overexpression of wild‐type Rap1b (to be used as a control). Using the modified DAMI cell lines we are beginning to understand the impact of Rap1b phosphorylation on proliferation on in MTS survival assays. Changes in cell survival can be attributed to changes in cell cycle progression and/or susceptibility to apoptosis. To address possible changes in cell cycle progression, we have evaluated each cell line based on the percentage of cell undergoing DNA synthesis at a given time by BrdU incorporation assays. To evaluate apoptosis susceptibility, mitochondrial membrane integrity and activation/cleavage of Caspase 9 was determined in the presence or absence of the apoptotic inducer ABT‐737. The analysis of the role of Rap1b in megakaryocytes has the potential to increase our collective understanding of regulation of platelet count and could be a novel target to combat thrombocytopenia (low platelet count) or thrombocythemia (high platelet count).Support or Funding InformationUniversity of Wisconsin ‐LaCrosse Faculty Research Grant

Mesenchymal stromal cells lower platelet activation and assist in platelet formation in vitro.

The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.

Genetic diversity and molecular characterization of Babesia motasi-like in small ruminants and ixodid ticks from China

Ovine babesioses, an important tick-borne disease of sheep and goats in China, is caused by the reproduction of intraerythrocytic protozoa of the Babesia genus. Babesia motasi-like is a Babesia parasite that infects small ruminant in China, and two sub-groups of B. motasi-like can be subdivided based on differences in the rhoptry-associated-protein-1 gene. This study aimed to characterize the distribution, epidemiology and genetics of B. motasi-like in animals and ticks. A molecular investigation was carried out from 2009 to 2015 in 16 provinces in China. In total, 1081 blood samples were collected from sheep and goats originating from 27 different regions, and 778 ixodid tick samples were collected from 8 regions; the samples were tested for the presence of B. motasi-like using a specific nested PCR assay based on the rap-1b gene. The results indicated that 139 (12.9%), 91 (8.4%), 48 (4.4%) and 6 (0.7%) of the blood samples were positive for general B. motasi-like, Babesia sp. BQ1 (Lintan and Ningxian), Babesia sp. Tianzhu and Babesia sp. Hebei sub-groups, mixed infections, respectively. Among the collected 778 ixodid ticks (including Haemaphysalis longicornis, Haemaphysalis qinghaiensis, Dermacentor silvarum, Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) microplus), the most frequently infected with Babesia were D. silvarum and I. persulcatus (35.7%), followed by H. longicornis (26.8%), H. qinghaiensis (24.8%) and R. sanguineus (9.3%). The PCR results were confirmed by DNA sequencing. The positive rates of B. motasi-like infection in ticks were found to be higher in China, compared with previous studies in other countries. B. motasi-like infections have not previously been reported in D. silvarum, I. persulcatus or R. sanguineus. The findings obtained in this study could be used for planning effective control strategies against babesiosis in China.

Characterization of RAP1A and RAP1B expression across cell types and identification of their core promoter elements (946.1)

The RAP1A and RAP1B genes are members of the RAS oncogene family of GTPases and are implicated in many processes, including cell adhesion, growth, and signal transduction. Cell type‐specific expression of these genes and their regulatory elements, however, have been poorly investigated. Our lab has worked to define the levels of RAP1A and RAP1B native expression in a variety of human cell lines and to characterize the cis‐regulatory elements controlling this expression. In investigating native expression, RNA was isolated from eight different lines and analyzed using qRT‐PCR. Our findings suggest that RAP1A is expressed at statistically similar levels in all lines tested, while RAP1B shows more variable expression. Putative promoter fragments for both genes were identified using the UCSC genome browser, cloned by PCR, and inserted directly upstream of the Green Fluorescent Protein (GFP) gene. Plasmids were transiently transfected into a variety of cell lines, and GFP expression was analyzed by fluorescence microscopy. Activity of the core promoter for RAP1A appears to explain the observed native expression, and a possible repressor element has been identified. For RAP1B, the identified core promoter fails to explain cell type‐specific native expression, requiring further analysis of potential enhancer regions. Additionally, lack of RAP1B core promoter function in an osteosarcoma line may be SP1‐mediated.

Features of the RAP1A and RAP1B core promoters: recombinant approaches to testing regulatory element function in expression control (946.10)

The RAP1 genes, closely related to the RAS oncogenes, have been shown to have numerous functions, yet little is known about the expression regulation of these genes in human cells. Our lab has constructed plasmids containing putative promoter regions of human RAP1A and RAP1B, driving a gene for green fluorescent protein (GFP). We have transiently transfected these plasmids into a wide‐range of human cell types, and analyzed GFP expression via fluorescence microscopy, qRT‐PCR, and western blotting. By comparing the efficacy of each putative core promoter segment, some distinct regulatory regions have been revealed. Additionally, comparing promoter‐driven expression to the actual native expression of the RAP1B genes in specific cell types has suggested an important regulatory role for enhancer elements not included in the core promoter regions. Various sub‐cloning techniques, including inverse fusion PCR cloning (IFPC), have been employed to add putative regulatory regions to existing GFP core promoter plasmids. These should allow us to further characterize the expression control of the RAP1 genes, and to understand the cell‐type differences observed in native RAP1 expression.

Rap1 Ameliorates Renal Tubular Injury in Diabetic Nephropathy

Rap1b ameliorates high glucose (HG)-induced mitochondrial dysfunction in tubular cells. However, its role and precise mechanism in diabetic nephropathy (DN) in vivo remain unclear. We hypothesize that Rap1 plays a protective role in tubular damage of DN by modulating primarily the mitochondria-derived oxidative stress. The role and precise mechanisms of Rap1b on mitochondrial dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound microbubble-mediated technique as well as in renal proximal epithelial tubular cell line (HK-2) exposed to HG ambiance. The results showed that Rap1b expression decreased significantly in tubules of renal biopsies from patients with DN. Overexpression of a constitutively active Rap1b G12V notably ameliorated renal tubular mitochondrial dysfunction, oxidative stress, and apoptosis in the kidneys of STZ-induced rats, which was accompanied with increased expression of transcription factor C/EBP-β and PGC-1α. Furthermore, Rap1b G12V also decreased phosphorylation of Drp-1, a key mitochondrial fission protein, while boosting the expression of genes related to mitochondrial biogenesis and antioxidants in HK-2 cells induced by HG. These effects were imitated by transfection with C/EBP-β or PGC-1α short interfering RNA. In addition, Rap1b could modulate C/EBP-β binding to the endogenous PGC-1α promoter and the interaction between PGC-1α and catalase or mitochondrial superoxide dismutase, indicating that Rap1b ameliorates tubular injury and slows the progression of DN by modulation of mitochondrial dysfunction via C/EBP-β–PGC-1α signaling.

P-0003 - Rnai-Mediated Silencing of Rap1B Gene Expression in Esophageal Squamous Cell Carcinoma Inhibits Tumor Growth and Invasion

Background: Rap1b is known to play a role in the progression of angiogenesis and migration. Our previous study found that Rap1b was over-expression in esophageal squamous cell carcinoma (ESCC). Based on our previous results, we investigated the role of Nrf2 in cell proliferation and invasion in ESCC.

Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes

Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LβT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40–60% using a sedimentation gradient. Given the limited number of gonadotropes, RNA was amplified prior to microarray analysis. Thirty-three genes were up-regulated 40min after GnRH stimulation. Real-time PCR confirmed regulation of several transcripts including fosB, c-fos, egr-2 and rap1b, a small GTPase and member of the Ras family. GnRH stimulated rap1b gene expression in gonadotropes, measured by a sensitive single cell assay. Immunocytochemistry revealed increased Rap1 protein in GnRH-stimulated gonadotropes. These data establish rap1b as a novel gene rapidly induced by GnRH and a candidate to modulate gonadotropin secretion in rat gonadotropes.

The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,

Abstract 3413Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown.Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish. Disclosures:No relevant conflicts of interest to declare.

Reduced Rap1 Signaling Is Associated with Prostate Cancer Progression, Migration, Invasion, and Metastasis

Rap1 is a small G‐protein regulator of E‐cadherin cell‐cell junctions and integrin‐mediated cell‐matrix adhesions. Our expression profiling of clinical prostate cancer tissue samples showed significantly decreased RAP1A mRNA levels in tumor compared to normal tissue samples, while a negative regulator of RAP1, RAP1GAP, was significantly increased. Furthermore, these changes in RAP1A and RAP1GAP expression are significantly correlated with Gleason tumor grade, suggesting that reduced RAP1 signals play a role in metastatic progression. We have analyzed RAP1GAP protein expression by immunohistochemistry using a tissue microarray with 139 cores from 79 prostate cancer patients, and observed that RAP1GAP staining intensity was significantly correlated with tumor grade (p < 0.001). We also show that constitutively active Rap1 signaling slows prostate cancer cell migration and invasion in vitro, while inhibition of Rap1 signaling accelerates cell migration and matrigel invasion. The PTEN‐PI3K pathway interacts with RAP1 signaling, but the mechanism is not well understood. Here we present data showing that the PTEN‐PI3K pathway is an important regulator of RAP1A and RAP1B gene expression.

Abstract 3662: Involvement Of The Small Gtpase Rap1 In Integrin Signaling In Endothelial Cells And Postnatal Neovascularization

Ras associated protein 1 (Rap1), a small GTPase of the Ras family, has attracted much attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes for integrin signaling in endothelial cells (EC) and angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVEC) express Rap1a and Rap1b mRNA as assessed by RT-PCR. In order to determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase activating protein, which specifically inhibits the activity of both Rap1a and Rap1b. Overexpression of Rap1GAP1 led to a significant inhibition of angiogenic sprouting of HUVEC under basal conditions and bFGF stimulation by 44 ± 5 % in a 3-dimensional spheroidal system and blocked tube formation in a matrigel assay, migration and adhesion. In order to separately investigate the role of Rap1a and Rap1b genes in angiogenesis, we performed gene silencing with siRNA. Silencing of either Rap1a or Rap1b significantly and additively blocked the sprouting of HUVEC under basal and bFGF-stimulated conditions (Rap1a-siRNA: 55 ± 5 %, Rap1b-siRNA: 61 ± 9 % and Rap1a+Rap1b siRNA: 73 ± 5% inhibition) and significantly reduced HUVEC migration and adhesion on fibronectin and collagen. Moreover, silencing of Rap1a and Rap1b reduced beta1-integrin affinity in HUVEC, suggesting the importance of Rap1a and Rap1b for inside-out integrin activation in EC. In addition, silencing of Rap1a and Rap1b prevented VEGF-induced PKB/Akt1 activation. These data prompted us to investigate the in vivo role of Rap1a using Rap1a-deficient mice. Interestingly, Rap1a −/− mice are born with a substantially reduced mendelian ratio. Rap1a +/− heterozygote mice displayed decreased microvessel density in comparison to wild-type mice (Rap1a +/+ ) in a matrigel plug assay. Moreover Rap1a +/− and Rap1a −/− displayed significantly reduced microvessel density in ischemic muscles in the model of hind limb ischemia in comparison to wild-type mice (Rap1a +/− : 32 ± 3 % ; Rap1a −/− : 43 ± 3 % inhibition). Thus, our data demonstrated a critical role of Rap1 in the regulation of β1-integrin signaling in endothelial cells and for postnatal neovascularization.

Mutation Analysis of RAP1 Gene in Papillary Thyroid Carcinomas

In human papillary thyroid carcinomas (PTCs), the genetic alterations of RET/PTC, RAS or BRAF account for about 60-70% of cases with practically no overlap, providing strong genetic evidence that constitutive active signaling along MAPK pathway is critical for PTC development. In the remaining 30-40% of the cases, the oncogenes are still unknown. RAP1 is a member of the RAS family of small G proteins transmitting signals from the TSH-R to MAPK pathway using cAMP-dependent mechanism in thyroid cells. RAP1 was shown to have both mitogenic and tumorigenic properties in certain systems; however, the potential role of RAP1 mutation in thyroid carcinogenesis has yet to be elucidated. In this study, we analyzed the mutational status of RAP1 gene in 36 Russian patients with PTCs without RET/PTC rearrangement, BRAF mutation or RAS mutation. No mutations in either RAP1A or RAP1B genes were found. These results suggest that RAP1 mutation does not play an important role in PTC pathogenesis.

Folate deficiency induced hyperhomocysteinemia changes the expression of thrombosis-related genes

Hyperhomocysteinemia (HH) is an independent risk factor for thrombosis although the precise pathogenesis is still unresolved. Previous studies have demonstrated that HH changes whole blood coagulation by increasing the velocity, increasing the firmness of the formed clot, and by prolonging the initiation phase of the coagulation. With the aim of elucidating the genetic pathogenesis which might be responsible for the changes in whole blood coagulation, we applied oligo-array technology to RNA from buffycoat-cells comparing animals suffering from hyperhomocysteinemia (42 micromol/l) with controls (6 micromol/l). Data mining identified a number of relevant genes, and the expression pattern was validated by real time reverse transcriptase-polymerase chain reaction. An upregulation of integrin beta-3, Rap 1b, glycoprotein V, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) led us to deduce increased platelet activation/aggregation. Coagulation factor XIIIa was upregulated and may contribute in increasing the firmness of the formed clot. Impaired fibrinolysis was anticipated, since an upregulation of plasminogen activator inhibitor-1 (PAI-1) and a downregulation of tissue-type plasminogen activator (t-PA) were detected. Reduced spontaneous contact activation was anticipated due to a downregulation of the kallikrein gene. Upregulation of selectins may contribute to increased tethering and rolling of leukocytes. In conclusion, folate deficiency induced hyperhomocysteinemia changes in the gene expression of buffy coat cells which was characterized by increased platelet activation, impaired fibrinolysis and a reduced contact activation of the coagulation. These changes may contribute to explain the increased risk of thrombosis seen in hyperhomocysteinemia individuals. This pattern of the hyperhomocysteinemia-affected genes may represent a reference for further studies at the protein level to define the folate depletion effects in blood cells.

Rap1A-Deficient T and B Cells Show Impaired Integrin-Mediated Cell Adhesion

Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1-/- cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.