von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.

Background: Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. Objectives: To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. Methods: VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. Results: We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. Conclusion: VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Impact of intraoperative use of venovenous extracorporeal membrane oxygenation on the status of von Willebrand factor large multimers during single lung transplantation.

Background: von Willebrand factors (vWFs), hemostatic factors, are produced as large multimers and are shear stress-dependently cleaved to become the appropriate size. A reduction in vWF large multimers develops in various conditions including the use of extracorporeal life support, which can cause excessive-high shear stress in the blood flow and result in hemostatic disorders. The objective of this prospective study was to investigate the impact of venovenous extracorporeal membrane oxygenation (VV ECMO) use on the status of vWF large multimers and hemostatic disorders during single lung transplantation (SLT). Methods: We prospectively enrolled 12 patients who underwent SLT at our center. Among them, seven patients were supported by VV ECMO intraoperatively (ECMO group) and the remaining five patients underwent SLT without ECMO support (control group). The vWF large multimer index (%) was defined as the ratio of the large multimer proportion in total vWF (vWF large multimer ratio) derived from a patient's plasma to that from standard human plasma. Results: The vWF large multimer index at the end of the surgery was significantly lower in the ECMO group than in the control group (112.6% vs. 75.8%, respectively; P<0.05). The intraoperative blood loss and the amounts of intraoperative transfusion products in the ECMO group tended to be greater than those in the control group; however, the differences were not significant. Conclusions: During SLT, the use of VV ECMO caused a decrease in the vWF large multimer index. The short duration of time of VV ECMO use in our study did not significantly affect the intra- and postoperative outcomes including blood loss, blood transfusion, and re-exploration thoracotomy for bleeding. Nevertheless, to comprehensively evaluate the actual influence of this decrease in the vWF large multimer index on intra- and postoperative outcomes, a multicenter larger-scale study is warranted.

Ral GTPase promotes metastasis of pancreatic ductal adenocarcinoma via elevation of TGF-ß1 production.

Pancreatic ductal adenocarcinoma (PDAC), caused by activating mutations in K-Ras, is an aggressive malignancy due to its early invasion and metastasis. Ral GTPases are activated downstream of Ras and play a crucial role in the development and progression of PDAC. However, the underlying mechanisms remain unclear. In this study, we investigated the mechanism of Ral-induced invasion and metastasis of PDAC cells using RalGAPß-deficient PDAC cells with highly activated Ral GTPases. Array analysis and ELISA revealed increased expression and secretion of TGF-ß1 in RalGAPß-deficient PDAC cells compared to control cells. Blockade of TGF-ß1 signaling suppressed RalGAPß deficiency-enhanced migration and invasion in vitro and metastasis in vivo to levels similar to controls. Phosphorylation of c-Jun N-terminal kinase, a repressor of TGF-ß1 expression, was decreased by RalGAPß deficiency. These results indicate that Ral contributes to invasion and metastasis of PDAC cells by elevating autocrine TGF-ß1 signaling at least in part by decreasing c-Jun N-terminal kinase activity.

Platelet αIIbß3 integrin binds to SARS-CoV-2 spike protein of alpha strain but not wild type and omicron strains.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a pandemic infectious disease, Coronavirus disease 2019 (COVID-19). It causes respiratory infection. Then, it progresses into a systemic infection by involving other organs. This progression mechanism remains to be elucidated, although thrombus formation plays an important role in its progression. Platelets is involved in the thrombus formation by aggregating each other through association of activated αIIbß3 integrin with the Arg-Gly-Asp (RGD) motif-containing its ligands such as fibrinogen and von Willebrand factor. SARS-CoV-2 enters host cells through association of the spike protein (S-protein) with its receptor, angiotensin-converting enzyme 2 (ACE-2), on the host cells. While presence of ACE2 in platelets is suspicious, S-protein harbors the RGD sequences within its receptor binding domain. Therefore, it could be possible SARS-CoV-2 enter platelets through association of S-protein with αIIbß3. In this study, we found that receptor binding domain of S-protein of WT SARS-CoV-2 strain barely bound to isolated healthy human platelets. In contrast, highly toxic alpha-strain-based N501Y substitution strongly bound to platelets in a RGD dependent manner, although binding of S protein did not induce platelet aggregation or activation. This binding may serve for transferring the infection to systemic organs.

Jejunal Angiodysplasia in an Elderly Patient with Aortic Stenosis: Significance of Von Willebrand Factor as an Etiologic Factor.

Heyde's syndrome is a disease in which patients with aortic stenosis (AS) bleed from angiodysplasia. An 80-year-old woman with a history of severe AS was referred to our hospital with melena and anemia. The patient underwent jejunal resection after repeated blood transfusions. A pathological examination revealed angiodysplasia, and the patient's plasma lacked high-molecular-weight von Willebrand factor (VWF) multimers, leading to the diagnosis of Heyde's syndrome. The patient underwent transcatheter aortic valve implantation (TAVI) one year after the diagnosis, and the VWF index recovered. This is a valuable case in which the pathological analysis of angiodysplasia associated with Heyde's syndrome was possible.

COVID-19 and Thrombosis: Clinical Aspects.

In coronavirus disease 2019 (COVID-19), thrombus formation is related to the pathogenesis of acute respiratory distress syndrome (ARDS) and the progression of clinical symptoms. Severe damage to vascular endothelial cells and the associated cytokine storm after SARS-CoV-2 infection cause thrombogenesis and contribute to the development of more severe and unique thromboses compared to other infectious diseases. Thromboses occur more often in critically ill patients. In addition to pulmonary thromboembolism (PE) and deep vein thrombosis, acute myocardial infarction, peripheral arterial thrombosis, and aortic thrombosis have also been reported. In PE, thrombi develop in both pulmonary arteries and alveolar capillaries. These, together with intraalveolar fibrin deposition, interfere with effective gaseous exchange in the lungs and exacerbate the clinical symptoms of ARDS in patients with COVID-19. Pharmacological thromboprophylaxis is recommended for all hospitalized patients to prevent both thrombosis and aggravation of ARDS, and other organ failures. Although the pediatric population is mostly asymptomatic or develops mild disease after SARS-CoV-2 infection, a new inflammatory disorder affecting the cardiovascular system, multisystem inflammatory syndrome in children (MIS-C), has been reported. Similar to Kawasaki disease, acute myocarditis, coronary vasculitis, and aneurysms are typically seen in MISC, although these two are now considered distinct entities. A similar acute myocarditis is also observed in young male adults, in which a hyperinflammatory state after SARS-CoV-2 infection seems to be involved. Several side effects following vaccination against COVID-19 have been reported, including vaccine-induced immune thrombotic thrombocytopenia and acute myocarditis. Although these could be serious and life-threatening, the cases are very rare, thus, the benefits of immunization still outweigh the risks.

Time of Day of Vaccination Does Not Associate With SARS-CoV-2 Antibody Titer Following First Dose of mRNA COVID-19 Vaccine.

The immune system exhibits circadian rhythms, and its response to viral infection is influenced by the circadian clock system. Previous studies have reported associations between the time of day of vaccination against COVID-19 and production of anti-SARS-CoV-2 antibody titer. We examined the effect of vaccination time of day on anti-SARS-CoV-2 antibody titer after the first dose of vaccination with the mRNA-1273 (Moderna) COVID-19 vaccine in an adult population. A total of 332 Japanese adults participated in the present study. All participants were not infected with SARS-CoV-2 and had already received the first dose of mRNA-1273 2 to 4 weeks prior to participating in the study. The participants were asked to provide basic demographic characteristics (age, sex, medical history, allergy, medication, and mean sleep duration), the number of days after the first dose of vaccination, and the time of day of vaccination. Blood was collected from the participants, and SARS-CoV-2 antibody titers were measured. Ordinary least square regression was used for assessing the relationship between basic demographic characteristics, number of days after vaccination, time of day of vaccination, and the log10-transformed normalized antibody titer. The least square mean of antibody titers was not associated with the vaccination time and sleep durations. The least square means of antibody titers was associated with age; the antibody titers decreased in people aged 50 to 59 years and 60 to 64 years. The present findings demonstrate that the vaccination time with mRNA-1273 was not associated with the SARS-CoV-2 antibody titer in an adult population, suggesting that these results do not support restricting vaccination to a particular time of day. The present findings may be useful in optimizing SARS-CoV-2 vaccination strategies.

Individual Variability in von Willebrand Factor Fragility in Response to Shear Stress: A Possible Clue for Predicting Bleeding Risk.

Acquired von Willebrand syndrome (AVWS), characterized by reduced von Willebrand factor (VWF) large multimers, has recently been implicated as the principal mechanism underlying bleeding in patients implanted with left ventricular assist devices (LVADs). Hematological severity of AVWS varies among patients, even if an identical device is implanted. We investigated whether this diversity in hematological severity is due to individual variability in VWF fragility, according to responses to incremental shear stress. Whole-blood samples were sheared at 20,000-40,000 s -1 shear rate, an index of shear stress, using a custom-made shear stressor that could generate shear stress compatible with that produced by an LVAD. The degree of VWF large multimers degradation was evaluated using the VWF large multimer index. A significant inverse correlation was observed between the VWF large multimer index and LVAD-compatible magnitudes of shear stress: the VWF large multimer indices were 68.5 ± 18.3, 48.0 ± 13.9, 33.9 ± 12.1, 23.7 ± 7.9, and 18.7% ± 8.7% at 20,000, 25,000, 30,000, 35,000, and 40,000 s -1 of shear rates, respectively ( P < 0.0001). Furthermore, experimental VWF large multimer index values were compatible with those derived from patients with implanted LVADs (median; 28.9%). Finally, reduction in the VWF large multimer index corresponding to shear stress showed individual variation. We demonstrated that the combined use of a novel high shear stress loading device and quantitative evaluation of VWF large multimers may predict risk of bleeding before LVAD implantation.

Ral GTPase-activating protein regulates the malignancy of pancreatic ductal adenocarcinoma.

The small GTPases RalA and RalB are members of the Ras family and activated downstream of Ras. Ral proteins are found in GTP-bound active and GDP-bound inactive forms. The activation process is executed by guanine nucleotide exchange factors, while inactivation is mediated by GTPase-activating proteins (GAPs). RalGAPs are complexes that consist of a catalytic α1 or α2 subunit together with a common ß subunit. Several reports implicate the importance of Ral in pancreatic ductal adenocarcinoma (PDAC). However, there are few reports on the relationship between levels of RalGAP expression and malignancy in PDAC. We generated RalGAPß-deficient PDAC cells by CRISPR-Cas9 genome editing to investigate how increased Ral activity affects malignant phenotypes of PDAC cells. RalGAPß-deficient PDAC cells exhibited several-fold higher Ral activity relative to control cells. They had a high migratory and invasive capacity. The RalGAPß-deficient cells grew more rapidly than control cells when injected subcutaneously into nude mice. When injected into the spleen, the RalGAPß-deficient cells formed larger splenic tumors with more liver metastases, and unlike controls, they disseminated into the abdominal cavity. These results indicate that RalGAPß deficiency in PDAC cells contributes to high activities of RalA and RalB, leading to enhanced cell migration and invasion in vitro, and tumor growth and metastasis in vivo.

COVID-19-Related Thrombosis in Japan: Final Report of a Questionnaire-Based Survey in 2020.

A questionnaire on COVID-19-related thrombosis in patients hospitalized before Aug 31, 2020, was sent to 399 hospitals throughout Japan. Responses were received from 111 (27.8%) with information on 6,202 COVID-19 patients. Of these, 333 and 56 required ventilation or extracorporeal membrane oxygenation (ECMO), respectively, and 212 died (3.4%). D-dimer levels were measured in 75.0% of the patients, revealing that 9.2% and 7.6% exhibited D-dimer increases of 3-8-fold and ≥8-fold the reference value, respectively. Thrombotic events occurred in 108 patients (1.86% of the 5,807 patients with available data) including symptomatic cerebral infarction in 24, myocardial infarction in 7, deep vein thrombosis in 41, pulmonary thromboembolism in 30, and other thrombotic events in 22. Some patients developed multiple thrombotic events. Thrombosis occurred in 32 patients with mild or moderate COVID-19 severity (0.59% of those with data available) and in 52 patients on ventilation or ECMO (13.5% of severe patients for whom data were available). Thrombosis occurred in 67 patients during worsening clinical condition and in 26 during recovery. Anticoagulant therapy was provided to 893 patients (14.6% of the 6,119 patients with available data), the main reasons being provided as elevated D-dimer levels and worsening clinical condition.

Hemolysis and von Willebrand factor degradation in mechanical shuttle shear flow tester.

Chronic blood trauma caused by the shear stresses generated by mechanical circulatory support (MCS) systems is one of the major concerns to be considered during the development of ventricular assist devices. Large multimers with high-molecular-weight von Willebrand factor (VWF) are extended by the fluid forces in a shear flow and are cleaved by ADAMTS13. Since the mechanical revolving motions in artificial MCSs induce cleavage in large VWF multimers, nonsurgical bleeding associated with the MCS is likely to occur after mechanical hemodynamic support. In this study, the shear stress (~ 600 Pa) and exposure time related to hemolysis and VWF degradation were investigated using a newly designed mechanical shuttle shear flow tester. The device consisted of a pair of cylinders facing the test section of a small-sized pipe; both the cylinders were connected to composite mechanical heads with a sliding-sleeve structure for axial separation during the withdrawing motion. The influence of exposure time, in terms of the number of stress cycles, on hemolysis and VWF degradation was confirmed using fresh goat blood, and the differences in the rates of dissipation of the multimers were established. The plasma-free hemoglobin levels showed a logarithmic increase corresponding to the number of cycles, and the dissipation of large VWF multimers occurred within a few seconds under high shear stress flow conditions.

Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking.

Ykt6 is an evolutionarily conserved SNARE protein regulating Golgi membrane fusion and other diverse membrane trafficking pathways. Unlike most SNARE proteins, Ykt6 lacks a transmembrane domain but instead has a tandem cysteine motif at the C-terminus. Recently, we have demonstrated that Ykt6 undergoes double prenylation at the C-terminal two cysteines first by farnesyltransferase and then by a newly identified protein prenyltransferase named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of a novel α subunit prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of Rab geranylgeranyltransferase. PTAR1 knockout (KO) cells, where Ykt6 is singly prenylated with a farnesyl moiety, exhibit structural and functional abnormalities in the Golgi apparatus with delayed intra-Golgi trafficking and impaired protein glycosylation. It remains unclear whether the second prenylation of Ykt6 is required for proper trafficking of lysosomal hydrolases from Golgi to lysosomes. Here, we show that lysosomal hydrolases, cathepsin D and ß-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space in PTAR1 KO cells. Moreover, maturation of these hydrolases was disturbed. LC3B, an autophagy marker, was accumulated in PTAR1 KO cells, suggesting defects in cellular degradation pathways. Thus, doubly prenylated Ykt6, but not singly prenylated Ykt6, is critical for the efficient sorting and trafficking of acid hydrolases to lysosomes.

Detection of acquired von Willebrand syndrome after ventricular assist device by total thrombus-formation analysis system.

AIMS: Bleeding is a serious complication in patients with continuous-flow left ventricular assist device (CF-LVAD). Acquired von Willebrand syndrome (AVWS; type 2A) develops because of high shear stress inside the pumps and is a cause of bleeding complication. Although von Willebrand factor (vWF) multimer analysis is useful for diagnosing AVWS, it is only performed in specialized research institutes. A novel microchip flow chamber system, the total thrombus-formation analysis system (T-TAS), is a point-of-care system to evaluate the thrombus-formation process and useful for monitoring platelet thrombus-formation capacity in patients receiving antiplatelet therapy and the diagnosis and evaluation of the clinical severity of von Willebrand disease type 1. However, little is known about the association between AVWS and platelet thrombus-formation capacity evaluated by T-TAS in patients with CF-LVAD. We aimed to evaluate the utility of T-TAS for easy detection of AVWS in patients with CF-LVAD. METHODS AND RESULTS: We simultaneously evaluated the vWF large multimers and T-TAS parameters in four consecutive patients with axial-type CF-LVAD and eight control patients treated with aspirin and warfarin. vWF large multimer index was defined as the proportion of large multimers in total vWF derived from a normal control plasma. T-TAS analyses different thrombus-formation processes using two microchips with different thrombogenic surfaces. PL24 -AUC10 levels in the platelet (PL) chip are highly sensitive for platelet functions, while AR10 -AUC30 levels in the atheroma (AR) chip allow the assessment of the overall haemostatic ability. vWF large multimer index and T-TAS parameters were decreased in all patients with CF-LVAD. The mean PL24 -AUC10 level (5.4 ± 2.9 vs. 219 ± 67; P < 0.01), AR10 -AUC30 level (338 ± 460 vs. 1604 ± 160; P < 0.01) and vWF large multimer index (49 ± 11% vs. 112 ± 27%; P < 0.01) were significantly lower in the patients with CF-LVAD than in control patients. One patient showed changes in T-TAS levels before and after implantation of CF-LVAD. PL24 -AUC10 and AR10 -AUC30 levels decreased from 438.1 to 5.0 and from 1667.9 to 1134.3, respectively. CONCLUSIONS: In patients with CF-LVAD, the platelet thrombus-formation capacity was extremely impaired because of AVWS, and T-TAS parameters could detect the presence of AVWS. T-TAS can be used for easy detection of AVWS as a point-of-care testing. Further studies with a large sample size are needed to validate our results in several LVAD models and evaluate the prognostic value of bleeding complications and thromboembolism in patients with LVAD.

[Acquired von Willebrand syndrome: a hemostatic disorder frequently encountered in the fields of cardiology and gastroenterology].

von Willebrand disease is a genetic hemostatic disorder that is caused by the qualitative or quantitative dysfunction of the von Willebrand factor (VWF), which is involved in hemostasis. A similar dysfunction sometimes develops without mutations, known as acquired von Willebrand syndrome (AVWS)1) , which has been associated with lymphoproliferative diseases, myeloproliferative diseases, malignant tumors, and hypothyroidism. Recently, it was remarkably noted that cardiovascular diseases with high intravascular shear stress could cause AVWS2-4) . The incidence of cardiovascular disease-associated AVWS is exceptionally high, and we may encounter such patients in daily clinical settings. Further, special consideration is necessary for the treatment of bleeding since the cause of AVWS is based on the accelerated degradation of VWF.

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Disappearance of Angiodysplasia Following Transcatheter Aortic Valve Implantation in a Patient with Heyde's Syndrome: A Case Report and Review of the Literature.

An 83-year-old woman with severe aortic stenosis was admitted to our hospital due to heart failure with refractory anemia requiring blood transfusions. She had repetitive bleeding episodes from endoscopically proven angiodysplasia in the stomach. Moreover, she repeatedly underwent endoscopic argon plasma coagulation for hemostasis. Importantly, she had a deficiency of the high-molecular-weight (HMW) multimers of von Willebrand factor (VWF), and she was diagnosed with Heyde's syndrome.After she underwent transcatheter aortic valve implantation (TAVI), aortic valve area and mean left ventricular aorta pressure gradient improved. Notably, endoscopy showed cessation of bleeding at 10 days after TAVI and the disappearance of angiodysplasia at 4 months after TAVI. Even at 2 years after TAVI, follow-up endoscopy showed remaining free of angiodysplasia in the stomach. She experienced no episodes of anemia since TAVI procedure. Additionally, analysis of HMW multimers demonstrated immediate and lasting recovery after TAVI.Recovery of HMW multimers of VWF with cessation of gastrointestinal bleeding following aortic valve replacement has been previously reported in a patient diagnosed with Heyde's syndrome. To the best our knowledge, this is the first case to demonstrate that angiodysplasia disappears after TAVI for a long term with endoscopic images in a patient with Heyde's syndrome. Here, we summarized case reports of patients with Heyde's syndrome that required aortic valve intervention. Cessation of gastrointestinal bleeding and anemia after aortic valve intervention for severe aortic stenosis may be attributed not only to recovery of HMW multimers of VWF but also to the disappearance of angiodysplasia.

Down-regulation of RalGTPase-Activating Protein Promotes Colitis-Associated Cancer via NLRP3 Inflammasome Activation.

BACKGROUND & AIMS: Ral guanosine triphosphatase-activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC. METHODS: We used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction. RESULTS: Bone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1ß, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors. CONCLUSIONS: Ral activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.

Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis.

Ribosome biogenesis is essential for maintaining basic cellular activities although its mechanism is not fully understood. Inhibitor of growth 4 (ING4) is a member of ING family while its cellular functions remain controversial. Here, we identified several nucleolar proteins as novel ING4 interacting proteins. ING4 localized in the nucleus with strong accumulation in the nucleolus through its plant homeodomain, which is known to interact with histone trimethylated H3K4, commonly present in the promoter of active genes. ING4 deficient cells exhibited slower proliferation and the alteration in nucleolar structure with reduced rRNA transcription, which was rescued by exogenous expression of GFP-ING4 to the similar levels of wild type cells. In the ING4 deficient cells, histone H3K9 acetylation and the key rRNA transcription factor UBF at the promoter of rDNA were reduced, both of which were also recovered by exogenous GFP-ING4 expression. Thus, ING4 could positively regulate rRNA transcription through modulation of histone modifications at the rDNA promoter.

Downregulation of RalGTPase-activating protein promotes invasion of prostatic epithelial cells and progression from intraepithelial neoplasia to cancer during prostate carcinogenesis.

RalGTPase-activating protein (RalGAP) is an important negative regulator of small GTPases RalA/B that mediates various oncogenic signaling pathways in various cancers. Although the Ral pathway has been implicated in prostate cancer (PCa) development and progression, the significance of RalGAP in PCa has been largely unknown. We examined RalGAPα2 expression using immunohistochemistry on two independent tissue microarray sets. Both datasets demonstrated that the expression of RalGAPα2 was significantly downregulated in PCa tissues compared to adjacent benign prostatic epithelia. Silencing of RalGAPα2 by short hairpin RNA enhanced migration and invasion abilities of benign and malignant prostate epithelial cell lines without affecting cell proliferation. Exogenous expression of wild-type RalGAP, but not the GTPase-activating protein activity-deficient mutant of RalGAP, suppressed migration and invasion of multiple PCa cell lines and was phenocopied by pharmacological inhibition of RalA/B. Loss of Ralgapa2 promoted local microscopic invasion of prostatic intraepithelial neoplasia without affecting tumor growth in a Pten-deficient mouse model for prostate tumorigenesis. Our findings demonstrate the functional significance of RalGAP downregulation to promote invasion ability, which is a property necessary for prostate carcinogenesis. Thus, loss of RalGAP function has a distinct role in promoting progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma.