Overexpression of gene DEP domain containing 1 and its clinical prognostic significance in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly seen malignancies worldwide, yet its regulatory mechanisms still need to be further illuminated. Abundant evidence revealed that aberrant expression of cancer-related genes contributes to CRC progression. DEP domain containing 1 (DEPDC1) has been found to play a crucial role in the carcinogenesis and development of malignancies. Nevertheless, limited studies have been concerned with the role of DEPDC1 in CRC. This study aimed to investigate the relationship between DEPDC1 expression and CRC clinicopathological parameters. METHODS: Solid CRC tissues and adjacent noncancerous tissues (ANCTs) (n = 150) were chosen randomly to detect the mRNA expression levels of DEPDC1 by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Formalin-fixed, paraffin-embedded (FFPE) blocks of CRC tissues and ANCTs (n = 150) were acquired to examine DEPDC1 protein expression levels by immunohistochemistry (IHC). RESULTS: DEPDC1 was significantly overexpressed in CRC tissues than that in ANCTs (P < .05). High protein expression of DEPDC1 was associated with poorer TNM stage and recurrence (P < .001 and P = .003, respectively). Kaplan-Meier survival analysis showed significantly shorter overall survival (OS) and disease-free survival (DFS) in DEPDC1 protein high-expression group compared with low-expression group (P < .05). Univariate analysis demonstrated that DEPDC1 protein expression was correlated with DFS (P = .005) and OS (P = .006). Multivariate analysis revealed that the combination of DEPDC1 protein expression and TNM stage has statistical significance in CRC prognosis prediction (P = .024 and P = .009, respectively). CONCLUSIONS: DEPDC1 may act as a potential biomarker for CRC detection as well as a prognostic predictor concerning the survival of CRC patients.

ARHGAP29 expression may be a novel prognostic factor of cell proliferation and invasion in prostate cancer.

Yes­associated protein (YAP) is a transcription­coupling factor that plays a central role in the Hippo pathway, and its activation regulates cell proliferation and carcinogenesis. YAP activation has been reported in various malignancies, conferring tumors with migratory and invasive abilities. Several studies have suggested that YAP expression is closely associated with prostate cancer. Furthermore, YAP has been revealed to regulate destabilization of F­actin associated with the cytoskeleton via Rho GTPase­activating protein 29 (ARHGAP29), suggesting that ARHGAP29 is associated with cancer metastasis. In the present study, the functions of ARHGAP29 were examined in four prostate cancer cell lines (22Rv1, LNCaP, DU145 and PC­3) and it was revealed that upregulation of ARHGAP29 in LNCaP and DU145 cells with the lowest expression of ARHGAP29 promoted cell proliferation and invasion. Conversely, ARHGAP29 knockdown in PC­3 cells with its highest expression level significantly reduced cell proliferation and invasion. In addition, immunohistochemistry of specimens from 133 patients who underwent radical prostatectomy was performed to investigate the clinical association between ARHGAP29 expression and prognosis in prostate cancer patients. Multivariate analysis demonstrated that ARHGAP29 was an independent prognostic factor for biochemical progression­free survival (P=0.0123). These findings indicated that ARHGAP29 in prostate cancer may be a potential prognostic biomarker and therapeutic target.

AGAP1 regulates subcellular localization of FilGAP and control cancer cell invasion.

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) regulate membrane trafficking and actin cytoskeleton. The molecular mechanism of how Arf GAPs regulate actin cytoskeleton remains to be elucidated. We identified AGAP1, a subtype of Arf GAP, as a binding protein of FilGAP, a Rac-specific GAP, in mammalian cells. AGAP1 binds to C-terminus of FilGAP whereas FilGAP binds to N-terminus of AGAP1 containing GLD domain. FilGAP co-localized with AGAP1 at intracellular vesicles and targeting of FilGAP at the vesicles requires its interaction with AGAP1. Consistently, depletion of endogenous AGAP1 induced the accumulation of endogenous FilGAP into paxillin-positive focal adhesions and actin cytoskeletal structures. Knockdown of endogenous AGAP1 suppressed cell spreading on collagen and the suppression was released by depletion of endogenous FilGAP. Moreover, depletion of AGAP1 in MDA-MB-231 cells promoted cell invasion in extracellular matrices and depletion of FilGAP blocked the invasion. Taken together, the present study suggests that AGAP1 may regulate subcellular localization of FilGAP and control cell migration and invasion through interaction with FilGAP.

CXCL12 promotes human ovarian cancer cell invasion through suppressing ARHGAP10 expression.

The CXCL12/CXCR4 axis is strongly implicated as key determinant of tumor invasion and metastasis in ovarian cancer. However, little is known about the potential downstream signals of the CXCL12/CXCR4 axis that contribute to ovarian cancer cell invasion and metastasis. ARHGAP10, a member of Rho GTPase activating proteins is a potential tumor suppressor gene in ovarian cancer. In this study, a negative correlation between the protein levels of CXCL12, CXCR4, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR2) and ARHGAP10 was uncovered in ovarian cancer tissues and paired adjacent noncancerous tissues. CXCL12 stimulation reduced the expression of ARHGAP10. Furthermore, the pretreatment of CXCR4 inhibitor (AMD3100) or the vascular endothelial growth factor receptor-2 (VEGFR2) inhibitor (SU1498) abrogated the CXCL12-deduced expression of ARHGAP10. Finally, an in vitro functional assay revealed that CXCL12 did not stimulate ovarian cancer cell invasion when ARHGAP10 was overexpressed or when ovarian cancer cells were pre-treated with AMD3100 or SU1498. Knockdown of ARHGAP10 significantly suppressed the inhibitory effects of SU1498 on ovarian cancer cell invasion and lung metastasis. In summary, these findings suggest that CXCL12/CXCR4 promotes ovarian cancer cell invasion by suppressing ARHGAP10 expression, which is mediated by VEGF/VEGFR2 signaling.

ACAP4 interacts with CrkII to promote the recycling of integrin ß1.

ACAP4, a GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (ARF6), plays import roles in cell migration, cell polarity, vesicle trafficking and tumorigenesis. Similarly, the ubiquitously expressed adaptor protein CrkII functions in a wide range of cellular activities, including cell proliferation, T cell adhesion and activation, tumorigenesis, and bacterial pathogenesis. Here, we demonstrate that ACAP4 physically interacts with CrkII. Biochemical experiments revealed that ACAP4550-660 and the SH3N domain of CrkII are responsible for the interaction. Functional characterization showed that the interaction is required for the recruitment of ACAP4 to the plasma membrane where ACAP4 functions to regulate the recycling of the signal transducer integrin ß1. Thus, we suggest that the CrkII-ACAP4 complex may be involved in regulation of cell adhesion.

Tumor Suppressive Role of ARHGAP17 in Colon Cancer Through Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: A few Rho GTPase activating proteins (RhoGAPs) have been identified as tumor suppressors in a variety of human cancers. ARHGAP17, a member of RhoGAPs, has been reported to be involved in the maintenance of tight junction and epithelial barrier. The present study aimed to explore its expression in colon cancer and the possible function in colonic carcinogenesis. METHODS: The mRNA and protein expression was assessed by realtime PCR and immunoblotting, respectively. Cell Counting Kit-8 (CCK-8) and Transwell assays were performed to evaluate cell proliferation and invasion, respectively. RESULTS: We found that ARHGAP17 expression was obviously lower in colon cancer specimens than in normal colonic mucosa. ARHGAP17 expression was associated with tumor stage, size and differentiation. In vitro analysis demonstrated that ARHGAP17 overexpression inhibited cell growth and invasion of HCT-8 and HCT-116 cells. In addition, an in vivo experimental metastasis model showed that ARHGAP17 overexpression restricted cancer metastasis to the lung. Mechanically, we found that Wnt signaling contributed to the functions of ARHGAP17 in colon cancer cells. Gene set enrichment analysis (GSEA) in The Cancer Genome Atlas dataset showed that the Wnt signaling pathway was negatively associated with ARHGAP17 expression. The mRNA expression of ß-catenin (an important signaling transducer of canonical Wnt signaling) gene (CTNNB1) was negatively correlated with ARHGAP17 expression. Immunoblot analysis of downstream effectors of ß-catenin (c-Myc/p27 and MMP7) in ARHGAP17 overexpressing colon cancer cells and metastatic tumors within the lung also validated the GSEA result. ARHGAP17 overexpression increased the phosphorylation of glycogen synthetase kinase 3ß, and decreased ß-catenin nuclear localization and transcriptional activity. Furthermore, inhibition of Wnt signaling by Wnt Inhibitor Factor-1 (WIF-1) in HIEC cells with ARHGAP17 knockdown significantly attenuated the promotion effects of ARHGAP17 knockdown on cell proliferation, invasion and the activation of ß-catenin. CONCLUSION: these results suggest that ARHGAP17 might serve as a tumor suppressor in colon cancer progression and metastasis through Wnt/ß-catenin signaling pathway.

Prognostic role of XTP1/DEPDC1B and SDP35/DEPDC1A in high grade soft-tissue sarcomas.

BACKGROUND: The outcome of patients with metastatic soft tissue sarcoma (STS) remains unfavourable and new therapeutic strategies are needed. The aim of this study was to determine the role of RhoGAP, XTP1/DEPDC1B and SDP35/DEPDC1A, as possible prognostic markers, to be used to identify candidate patients for more effective and personalized therapies. MATERIALS-METHODS: SDP35/DEPDC1A and XTP1/DEPDC1B transcriptional levels were evaluated by Real-Time PCR in 86 primary STS and 22 paired lung metastasis. 17 normal tissues were used as control. Protein expression was evaluated by tissue microarray, including 152 paraffin-embedded STS samples and by western blot in 22 lung metastases and paired primary STS. Non-parametric and parametric analysis were used to establish the differences in gene and protein expression and prognostic factors were tested with Kaplan Meier and Cox's regression analyses. RESULTS: SDP35/DEPDC1A and XTP1/DEPDC1B gene were down-regulated in adjacent normal tissues while sarcoma specimens presented high mRNA levels, significantly related to metastasis-free survival. Gene expression further increased in paired metastatic lesions. Immunohistochemical staining showed a variable expression in intensity and distribution, with a significantly higher probability of metastatic disease in patients up-regulating SDP35/DEPDC1A. Western blotting assessed high levels of proteins in STS specimens and indicated a stronger expression of SDP35/DEPDC1A in metastases when compared to primary tumours. Multivariate analyses highlighted that SDP35/DEPDC1A abundance, grade III and no history of radiation therapy were significant independent risk factors. CONCLUSIONS: Our results demonstrated that increased expression of SDP35/DEPDC1A and XPT1/DEPDC1B correlates with metastatic progression and identified SDP35/DEPDC1A as an independent marker for prediction of poor prognosis.

ITRAQ-Based Proteomics Analysis of Acute Lung Injury Induced by Oleic Acid in Mice.

BACKGROUND/AIMS: This study was conducted to investigate the relationship between differentially expressed proteins (DEPs) and the pathogenesis of oleic acid (OA)-induced acute lung injury (ALI) in mice. METHODS: Eight-week-old male C57BL/6 mice were injected with OA through the tail vein and sacrificed 6 hours after OA administration to identify protein expression levels in lung tissue using isobaric tags for relative and absolute quantification (iTRAQ) technology. Then, DEPs such as antithrombin III (AT III), 12-lipoxygenase (12-LO), dedicator of cytokinesis 2 (DOCK2), polycystin-2 and plasminogen were identified by western blotting. Subsequently, we focused on investigating the effect of AT III on endothelial integrity using siRNA interference technology. The levels of IL-6, IL-1ß, TNF-α and TGF-ß expression were detected using an enzyme-linked immunosorbent assay (ELISA). Alterations in the tight junction component ZO-1 and the phosphorylation of myosin light chain (pMLC) were determined by western blotting. The stress fiber F-actin were also detected by immunofluorescence staining. In addition, endothelial permeability was determined via a transwell permeability assay. RESULTS: A total of 5152 proteins were found to be expressed in lung tissues from the OA-treated and saline-treated mice. Among these proteins, 849 were differentially expressed between the two groups, including 545 upregulated and 304 downregulated proteins. After AT III knockdown, the levels of inflammatory factors and endothelial permeability were elevated, the expression of ZO-1 was decreased, and the expression of F-actin and pMLC was increased. All these results illustrated that AT III knockdown exaggerated the disruption of endothelial integrity mediated by OA. CONCLUSION: These findings using iTRAQ technology demonstrate, for the first time, differences in the lung tissue expression levels of proteins between OA-treated mice and saline-treated mice. This study reveals that 12-LO, DOCK2 and especially AT III may be candidate biomarkers for OA-induced acute lung injury.

ACAP3, the GTPase-activating protein specific to the small GTPase Arf6, regulates neuronal migration in the developing cerebral cortex.

The GTPase-activating protein (GAP) specific to the small GTPase Arf6, ACAP3, is known to regulate morphogenesis of neurons in vitro. However, physiological significance of ACAP3 in the brain development in vivo remains unclear. Here, we show that ACAP3 is involved in neuronal migration in the developing cerebral cortex of mice. Knockdown of ACAP3 in the developing cortical neurons of mice in utero significantly abrogated neuronal migration in the cortical layer, which was restored by ectopic expression of wild type of ACAP3, but not by its GAP-inactive mutant. Furthermore, morphological changes of neurons during migration in the cortical layer were impeded in ACAP3-knocked-down cortical neurons. These results provide evidence that ACAP3 plays a crucial role in migration of cortical neurons by regulating their morphological change during development of cerebral cortex.

The Role of Tumor Suppressor DLC-1: Far From Clear.

BACKGROUND: Deleted in liver cancer 1 (DLC-1) In human was originally isolated from rats brain and was often found to be deleted in hepatocellular carcinoma (HCC). METHODS: We undertook a structured search of bibliographic databases for peer-reviewed research literature using a focused review question and inclusion/exclusion criteria. RESULTS: Subsequent studies have demonstrated that DLC-1 is generally expressed in normal human tissues as well as in rats, while it always exists inactivated or even lost in many human cancers, which characterizes DLC-1 as a potential tumor suppressor. Additionally, the RhoGAP (Rho-GTPase activating proteins) activity was found to play a pivotal role in regulating DLC-1. CONCLUSION: Although emerging studies in a variety of cancers have identified DLC-1 and its downstream signaling molecules as potential therapeutic targets for treatments of DLC-1-related cancers, the mechanisms linked to DLC-1 remain undefined.

Clinicopathological Significance of the Proliferation Markers Ki67, RacGAP1, and Topoisomerase 2 Alpha in Breast Cancer.

Objectives The aims of this study are to evaluate expressions of Ki67, RacGAP1 (MgcRacGAP) and topoisomerase 2 alpha (TOP2a), the markers related with cell proliferation that have been proposed to affect the prognosis in the literature and correlate the results with clinicopathological parameters of breast cancer patients. Methods Ki67, RacGAP1, and TOP2a antibodies were applied immunohistochemically to the tissue micrarray blocks of 457 female breast cancer patients. The results were correlated with clinical, prognostic, histopathological features, and other immunohistochemical findings (estrogen receptor [ER], progesterone receptor [PR], HER2, cytokeratin [CK]5/6, CK14, epidermal growth factor receptor [EGFR] and vimentin), statistically. Results Ki67 expression demonstrated direct correlation with TOP2a expression, mitotic count, tumor grade, geographic necrosis, basal-like phenotype. RacGAP1 expression was directly correlated with TOP2a expression, nipple invasion, and number of metastatic lymph nodes, and it was inversely correlated with PR expression. TOP2a expression was directly correlated with vimentin and Ki67 expressions, mitotic count, tumor grade, and geographic necrosis, and nipple invasion, and negatively correlated with ER and PR expressions. Higher TOP2a and Ki67 expressions were correlated with shorter overall survival. Higher TOP2a expression and RacGAP1 positivity were directly correlated with shorter disease-free survival. Conclusion This study showed that the overexpressions of Ki67, RacGAP1, and TOP2a affect the prognosis adversely, thus to develop target therapies against RacGAP1 and TOP2a as well as using Ki67 as a part of routine pathology practice might be beneficial in breast cancer therapy and prediction of prognosis.

DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers.

The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.

DLC-1 is an independent prognostic marker and potential therapeutic target in hepatocellular cancer.

BACKGROUND: The 5-year survival rate of patients with hepatocellular cancer (HCC) was very low because of invasion and metastasis in the early stage. Biomarkers might help predict early occurrence of invasion and metastasis. Accumulating evidence has shown that deleted in liver cancer-1 (DLC1) may be considered as a metastasis suppressor gene in numerous solid and hematological cancers. However, its prognostic role and mechanisms that regulate and coordinate these activities remain poorly understood. METHODS: With the method of immunohistochemistry, the expression of DLC-1 as well as Rho A, ROCK2, moesin had been characterized in 80 HCC tissues and adjacent noncancerous tissues. The correlation between their expression and their relationships with clinicopathological characteristics of HCC were also investigated. In addition, the prognostic value of DLC1 expression within the tumor tissues was assessed by Cox regression and Kaplan-Meier analysis. RESULTS: DLC1 expression was significantly lower in HCC tissues than in adjacent noncancerous tissues, and DLC-1 expression was found to be negatively correlated with tumor differentiation, TNM stage and lymph node metastasis. Furthermore, DLC-1 expression was found to inversely correlate with Rho A, ROCK2 and moesin which were all highly expressed in HCC tissues. Kaplan-Meier analysis showed that significantly longer 5-year survival rate was seen in HCC patients with higher DLC1 expression, compared to those with lower expression of DLC1. Multivariate Cox proportional hazard analyses revealed that DLC1 was an independent factor affecting the overall survival probability. CONCLUSION: DLC1 could be served as a tumor suppressor gene in the progression especially in the invasion and metastasis of HCC. DLC1 perhaps played its role by regulating the expression of Rho A, ROCK2 and moesin. Evaluation of the expression of DLC-1 might be a good prognostic marker for patients with HCC.

Opposing prognostic roles of nuclear and cytoplasmic RACGAP1 expression in colorectal cancer patients.

Rac GTPase activating protein 1 (RACGAP1) plays a regulatory role in initiation of cytokinesis, control of cell growth and differentiation, and tumor malignancy, making it a potential prognostic biomarker. RACGAP1 is present in the nucleus, but a diffuse distribution in the cytoplasm also occurs. The aim of this study was to determine the impact of nuclear and cytoplasmic expression of RACGAP1 on clinical outcome to provide further evidence of a role in colorectal cancer. RACGAP1 expression was analyzed by immunohistochemistry in 166 cancer specimens from primary colorectal cancer patients. The mean follow-up time after surgery was 5.4 years (range, 0.01-13.10 years). The prognostic value of RACGAP1 on overall survival was validated by Kaplan-Meier analysis and Cox regression models. RACGAP1 is expressed in colorectal specimen and is present in both the nucleus and cytoplasm in different amounts. Colorectal cancer patients had opposite prognoses depending on the site of RACGAP1 expression. Patients with high nuclear RACGAP1 expression had poor outcomes, whereas those with high cytoplasmic RACGAP1 expression had favorable prognosis (P = .003 and P = .001, respectively). Patients with low nuclear but high cytoplasmic RACGAP1 expression had better survival compared with those with other combinations (P < .001). We suggest that RACGAP1 expression levels in the nucleus and cytoplasm, determined by immunohistochemical staining, predict opposite clinical outcomes and that both could be independent prognostic markers for colorectal cancer.

A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities.

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg(2+) plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg(2+) concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z'-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add-mix-read, and applicable to high-throughput screening.

Nuclear GIT2 is an ATM substrate and promotes DNA repair.

Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses.

Proteomic profiling reveals autoimmune targets in sarcoidosis.

RATIONALE: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. OBJECTIVES: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. METHODS: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. MEASUREMENTS AND MAIN RESULTS: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. CONCLUSIONS: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

Nuclear envelope localization of Ran-binding protein 2 and Ran-GTPase-activating protein 1 in psoriatic epidermal keratinocytes.

The nuclear localization signal (NLS)-containing proteins LEDGF and STAT3 localize to the nucleus in both the spinous and basal layers of the epidermis in psoriatic skin, where they function as transcription factors or co-factors to activate epidermal keratinocytes (KCs). However, the mechanism underlying the localization of these proteins remains to be elucidated. We investigated the differential nucleocytoplasmic transport of NLS-containing proteins as a potential pathogenic mechanism for psoriasis vulgaris. Nucleoporins play an important role in the Ran-GTP-dependent nucleocytoplasmic transport of NLS-containing proteins. We showed, using immunohistochemical staining, that the nucleoporins Ran-binding protein 2 (RanBP2) and Ran-GTPase-activating protein 1 (RanGAP1) have greater expression on the nuclear envelope in psoriatic epidermal KCs than in KCs from healthy controls. We then studied the signalling pathways involved in the regulation of these proteins in HaCaT cells. The two major downstream pathways of epidermal growth factor receptor (EGFR) signalling activated in psoriatic KCs are the MAPK/Erk/1/2 and the phosphatidylinositol-3-kinase/Akt pathways. Therefore, we treated HaCaT cells with inhibitors to disrupt the MAP kinase kinase 1 (MEK1), PI3-kinase, or mTOR pathways. RanBP2 and RanGAP1 protein expression levels were significantly greater in the nuclear envelope of HaCaT cells that were not treated with inhibitors than in cells treated with a combination of PI3-kinase and MEK1 inhibitors or mTOR and MEK1 inhibitors. These results suggest that adequate nuclear envelope expression of RanBP2 and RanGAP1 could be a prerequisite for nucleocytoplasmic transport in KCs in psoriatic epidermis.

The scat mouse model highlights RASA3, a GTPase activating protein, as a key regulator of vertebrate erythropoiesis and megakaryopoiesis.

Although significant progress has been made in the past decades in our understanding of bone marrow failure syndromes and anemia, many pathological conditions of unknown origin remain. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). RASA3 is lost during reticulocyte maturation through the exosomal pathway and is therefore absent in mature erythrocytes. In wild-type reticulocytes, RASA3 is bound to the plasma membrane, a prerequisite for its GAP activity, but is mislocalized to the cytosol in scat. This mislocalization leads to RASA3 loss of function and higher levels of Ras-GTP, the active form of Ras, are consistently found in scat mature red cells. Finally, RASA3 function is conserved among vertebrates, since erythropoiesis and thrombopoiesis are impaired in zebrafish in which rasa3 is knocked-down by morpholinos, and RASA3 is expressed in human erythroleukemia cells as well as in primary cells. In this commentary, we highlight the critical, conserved and non-redundant function of RASA3 in the context of vertebrate erythropoiesis and megakaryopoiesis. We notably discuss the mechanism of RASA3 downregulation and speculate on the most intriguing part of the phenotype observed in scat; the transient remission period.